EPR STUDY OF LIGAND-RECEPTOR INTERACTIONS:

MEASURING LIGAND INDUCED CHANGES IN

DYNAMICS AND STRUCTURE OF THE ESTROGEN

RECEPTOR LIGAND BINDING DOMAIN

A Dissertation Presented

By

Stefano V. Gulla`

To

The Department of Chemistry and Chemical Biology

in partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

in the field of

Chemistry

Northeastern University Boston, Massachusetts

December 2008

2

EPR STUDY OF LIGAND-RECEPTOR INTERACTIONS:

MEASURING LIGAND INDUCED CHANGES IN

DYNAMICS AND STRUCTURE OF THE ESTROGEN

RECEPTOR LIGAND BINDING DOMAIN

By

Stefano V. Gulla`

ABSTRACT OF THESIS

Submitted in partial fulfillment of the requirements for the degree of Doctor of

Philosophy in Chemistry in the Department of Chemistry and Chemical Biology in

the Graduate School of Arts and Sciences of Northeastern University, Boston,

Massachusetts, December, 2008

3

Abstract

Ligand-receptor interactions are powerful determinants of biochemical

pathways. In this work, the role of ligand binding and coregulator interaction is

examined in particular as it related to changes in receptor dynamics and

conformation. The emerging technique of site directed spin labeling and electron

paramagnetic resonance (EPR) is applied to investigate ligand-induced effects on

estrogen receptor (ER), a pharmaceutically relevant member of the nuclear receptor

superfamily of transcription factors.

Chapter 1 introduces the relevant background information on the biology,

structure and physiology of NRs, with emphasis on the alpha isoform of the receptor

(ER-α). An overview of the current state of the field is presented. This chapter also

introduces application of EPR in the context of biological investigation of proteins

dynamics and structure

The specific techniques and methods used in this study are explored in detail

in chapter 2. Here the rationale of combining estradiol ligand substituted at the 11β

position with spin labeling of helix 12 of ER-α is discussed. This chapter provides

experimental details regarding ER mutant production and characterization. Relevant

details about EPR theory and lineshape analysis are explored. In this chapter, double

electron-electron resonance (DEER) is discussed by introducing both a theoretical

and experimental topics.

In chapter 3, the dynamic response of the human ER-α ligand binding

domain (ERα-LBD, residues 302-552) to the binding of different ligands and

4

coactivator peptides is investigated by site-directed spin labeling EPR (SDSL-EPR).

Specific labeling at residue 543 of the C-terminal helix 12 (H12) domain has

provided the first direct experimental demonstration that this domain undergoes

dynamic changes in response to ligand binding that correlate with the ligand’s

biological activity. Ligand-dependent changes are also observable for a label

positioned at residue 530 in the H12 hinge region, however much more dramatic

changes are observed at this position in the presence of both ligand and coregulator

peptides. We investigated the ligand/coregulator induced changes in structure by

measuring interspin distances in 530 labeled ERα-LBD dimers using DEER. These

results extend the current model of ERα-LBD action and provide dynamic

information on the H12 region as well as quantitative structural information on the

dimer conformation of ERα-LBD in solution. The results suggest a large-scale

remodeling of the ER dimer complex that is sensitive to details of the ligand structure

as well as the nature of the coactivator sequence.

In chapter 4 two nitroxide labeled estradiols, HO-2105 and HO-2447 are

characterized as new molecular probes of ligand/receptor interaction with the ERα-

LBD. EPR spectroscopy was used to investigate the binding properties and local

dynamics of the spin-labeled ligand. Fluorescence spectroscopy demonstrated

quenching of both the fluorescence of estradiol and of the intrinsic tryptophan

fluorescence of ERα-LBD by the nitroxide moiety of the labeled ligand. We describe

two methods to assay binding of the probes to ERα-LBD: (i) an EPR derived binding

assay and (ii) an intrinsic tryptophan fluorescence quenching binding assay.

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Saturation binding studies of the estrogen probes using the two assays showed good

agreement between the independent techniques. DEER spectroscopy was used to

measure interspin distances between the bound probes in the ERα-LBD homodimer

complex. The structural results are consistent with X-ray crystallography of the ERα-

LBD dimer and provide new information about the distribution of conformations in

the homodimer. The spin labeled estradiols described here serve as versatile probes of

ligand binding, local dynamics and structure with potential applications as ER

selective imaging agents and as oxidative stress probes.

Chapter 5 includes results obtained on other projects not related to ER, but all

sharing the broader theme of application of EPR to study biophysical systems. EPR is

a versatile technique that enables study of a variety of biophysical phenomena. Here

we describe applications of EPR as a method to evaluate singlet oxygen (1O2),

production, characterize conformational effects of hydrophobic mismatch on

transmembrane helices, develop methods for characterizing protein self-assembly and

characterize unstructured protein domains. Furthermore, as these projects result from

collaborations with research groups from different disciplines such as organic

chemistry, medicinal chemistry, biochemistry and mechanical engineering they each

add a particular set of challenges.

Chapter 6 evaluates future directions for continuing the investigation into the

molecular basis of ER action. Here we develop a theoretical model for the effect of

ligand/coregulator interaction on ER that is based on the combined results previously

presented. We also consider foreseeable challenges and provide recommendations

relative to experimental design on new spin labeled ER investigations.

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Table of contents

Chapter 1: Background and motivation __________________________________ 9 1.1 The Nuclear Receptor Superfamily _____________________________________ 10 1.2 ER-α Structure and Function__________________________________________ 13 1.3 Estrogen Receptor role in breast cancer _________________________________ 15 1.4 Site directed spin labeling and EPR: application to protein dynamics and structure ______________________________________________________________ 18

(a) Measuring local dynamics___________________________________________________ 18 (b) Measuring distance by EPR _________________________________________________ 22

1.5 Spectroscopy _______________________________________________________ 23 (a) CW-EPR ________________________________________________________________ 23 (b) Effect of molecular motion on EPR spectrum of nitroxides in solution ________________ 25 (c) DEER spectroscopy _______________________________________________________ 29

1.6 Overview___________________________________________________________ 32 Summary _____________________________________________________________ 36

Chapter 2: Methods _________________________________________________ 37 2.1 Study Design________________________________________________________ 38 2.2 Specific aims________________________________________________________ 39 2.3 Site directed mutation ________________________________________________ 40

(a) Selection of mutation sites and site directed mutagenesis___________________________ 40 (b) Expression, purification and spin labeling ______________________________________ 41 (c) Characterization of purified mutants by LC-MS and [3H]estradiol binding assay ________ 43

2.4 Pitfalls and optimization of experimental conditions _______________________ 50 Summary:_____________________________________________________________ 54

Chapter 3: Dynamic and structural response of the estrogen receptor ligand-binding domain to ligand and coactivator binding_________________________ 55

3.1 Introduction ________________________________________________________ 56 3.2 Materials and Methods _______________________________________________ 60

(a) Protein Expression, Purification and Spin Labeling _______________________________ 60 (b) Ligands _________________________________________________________________ 61 (c) Peptides _________________________________________________________________ 62 (d) Synthesis of IMSL_________________________________________________________ 63 (e) Mutant characterization_____________________________________________________ 64 (f) Incubation of labeled receptor with ligand/coactivators: ____________________________ 65 (g) CW-EPR spectroscopy _____________________________________________________ 65 (h) DEER Spectroscopy _______________________________________________________ 66

3.3 Results_____________________________________________________________ 67

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(a) Selection of labeling sites ___________________________________________________ 67 (b) Ligand induced dynamic changes of H12 _______________________________________ 68 (c) Dynamic and structural changes of H12 hinge region in response to ligand-coregulator interaction __________________________________________________________________ 71

3.4 Discussion __________________________________________________________ 79 3.5 Conclusions ________________________________________________________ 84

Chapter 4: Characterization of Spin labeled Estradiol _____________________ 86 Abstract ________________________________________ Error! Bookmark not defined. 4.1 Introduction ________________________________________________________ 87 4.2 Methods ___________________________________________________________ 88

(a) Protein preparation: ________________________________________________________ 88 (b) CW-EPR measurements:____________________________________________________ 89 (c) Fluorescence quenching assay: _______________________________________________ 89 (d) EPR assay _______________________________________________________________ 89 (e) DEER measurements: ______________________________________________________ 90

4.3 Results_____________________________________________________________ 91 (a) EPR characterization _______________________________________________________ 91 (b) EPR based binding assay____________________________________________________ 93 (c) Electronic absorption and emission properties ___________________________________ 95 (d) Fluorescence quenching binding assay _________________________________________ 96 (e) Distance measurement of ERα-LBD homodimer using DEER_______________________ 99

4.4 Discussion _________________________________________________________ 100 4.5 Conclusion ________________________________________________________ 102 Summary ____________________________________________________________ 103

Chapter 5: Other Projects ___________________________________________ 104 5.1 Development of an EPR spin trap assay to measure singlet oxygen sensitization of DNA binding dye compounds____________________________________________ 105

(a) Photodynamic therapy in cancer treatment _____________________________________ 105 (b) 1O2 specific spin trapping using TEMP________________________________________ 107

5.2 Effect of membrane thickness on cannabinoid receptor 1 transmembrane helical conformation _________________________________________________________ 113

(a) Introduction _____________________________________________________________ 113 (b) Experimental Procedures___________________________________________________ 116 (c) Results _________________________________________________________________ 120 (d) Discussion ______________________________________________________________ 131 (e) Conclusion______________________________________________________________ 137

5.3 Molecular-scale force measurements in a coiled-coil peptide by electron spin resonance ____________________________________________________________ 138 5.4 SDSL investigation into the structure of UmuD __________________________ 147

(a) Background _____________________________________________________________ 147

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(b) Initial results ____________________________________________________________ 148 (c) Ongoing work ___________________________________________________________ 151

Chapter 6: Ongoing and Future Work _________________________________ 152 6.1 Distance measurements in ER dimer___________________________________ 153

(a) Hypothesis of dimer remodeling _____________________________________________ 153 (b) Saturation recovery EPR ___________________________________________________ 156

6.2 Double labeled ER __________________________________________________ 157 (a) Experimental considerations ________________________________________________ 157 (b) Spin dilution ____________________________________________________________ 157 (c) Immobilization to substrate _________________________________________________ 158 (d) Initial results ____________________________________________________________ 159

6.3 Electrostatic actuation of leucine zipper peptide dimer____________________ 161 (a) Initial results and interpretations _____________________________________________ 161 (b) Difficulties with initial interpretation _________________________________________ 168

6.4 Initial characterization of 11β spin labeled estradiol ______________________ 171 6.5 Challenges ________________________________________________________ 174

(a) ER stability _____________________________________________________________ 174 (b) ER activity assay_________________________________________________________ 175

6.6 Broader Impact ____________________________________________________ 176 References ___________________________________________________________ 179

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Chapter 1: Background and motivation

10

1.1 The Nuclear Receptor Superfamily Members of the superfamily of nuclear receptors (NRs) are a class of intracellular

proteins whose general role is to mediate genomic transcription in response to ligand

activation. In other words, NRs translate a chemical signal, usually in the form of

lipophilic small molecules, to a biological response that is specific for each NR and

often dictated by tissue type. They respond directly to physical association with a

variety of hormonal or metabolic molecules and regulate the action of multiple

signaling cascades that regulate tissue-specific gene expression 1. More than 65 NRs

have been identified throughout the animal kingdom. Their structure is characterized

by a modular architecture of 5-6 conserved domains with independent functionality.

NRs are distinct from other transcription factor by their capacity to specifically bind

small hydrophobic ligands with high affinity and selectivity 2-4.

NR signaling is essential for normal homeostasis and proper tissue

development. Given the major impact that this class of proteins on cell physiology it

is not surprising that many pathologies are directly linked to NRs. Table 1

summarizes the link between NRs and some important disease states. Some endocrine

therapies have yielded successful treatments of NR related cancers such as breast

cancer, prostate cancer, acute promyelocytic leukemia (APL) and prevention of

certain types of cancers 5-12. Development of synthetic ligands with agonist,

antagonist, and partial-agonist properties as well as receptor-specific imaging has

created considerable interest in NRs as pharmaceutical targets. This effort has been

facilitated in large part by structural studies of the ligand binding domains (LBD) of

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numerous NRs which have provided important understanding of the molecular basis

of their biological function. More recently, understanding of NR binding partners

(coregulators) provides the basis for new strategies of pharmacological interference

with NR pathways. Although several NRs have been crystallized in the presence of

both agonist and antagonists, these static models have left many important questions

unanswered:

(i) How does ligand binding affects the dynamics and structure of the

receptors?

(ii) What is the effect of allosteric coregulator binding?

(iii) How does the interplay between agonist/antagonist ligands and

coregulators determine the active conformation of the receptor?

These questions are at the forefront of research in the field of NRs, but also signal the

need for techniques that go beyond X-ray crystallography and are capable of looking

at complex protein/ligand interaction in a more physiological environment. Recently

other methods have been used to address the important questions relative to the NR’s

ligand and coregulator interactions; these include fluorescence labeling, NMR, and

deuterium exchange mass spectrometry.

The work described here focuses on estrogen receptor (ER), one of the most

important NRs for human health. A major aim is to measure dynamic and structural

effects of ligand binding and coregulator interaction with ER as a way to gain new

insights into the pharmacology of ER and, more generally, of NRs. At the same time

this work evaluates new tools that are adapted to address the specific questions

regarding ER function. New estradiol derived ligands with potential application as

12

biophysical tools and imaging agents were designed and characterized. Furthermore,

solution structure constraints for ER-α were obtained for the first time.

13

Nuclear Receptor Ligand Associated Pathology hERα hERβ

OH

OH

Breast Cancer Prostate Cancer Osteoporosis

hPR hGR hMR hAR

Gestation, Abortion Inflammation Prostate cancer

hRARα1 hRARβ2 hRAR γ1 hRXRα hRXRβ hRXRγ

O

OH

Cancer prevention APL (Acute promyelocytic leukemia)

hPPARα

OHO

OH

OH

Inflammation

hTRα hTRβ

OH

O

NH2

I

OH

I

I

I

O

Obesity

hVitD3

OH

Bone Development

Figure 1.1: Overview of members of the superfamily of nuclear receptors. The receptors are grouped by ligand similarity (center) and their pharmacologic relevance is indicated (right).

1.2 ER-α Structure and Function Estrogen Receptor alpha (ER-α) is a transcription factor belonging to the nuclear

receptor (NR) superfamily that acts to regulate the expression of target genes

involved in development, metabolism and reproduction. ER-α transactivation is

estradiol dependent and it is directed to genomic estrogen response elements (ERE)

Estradiol

Progesterone

all-trans retinoic acid

Leukotriene B4

Thyroxine

Vitamin D3

14

by tissue specific co-activator and co-repressor proteins. Members of the NR family

share limited sequence similarities; however, they are structurally homologous with

functionally independent domains 13,14. This characteristic architecture makes it

possible to investigate the activity of individual domains separately. The full length

ER-α is composed of an N-terminal activation function 1 (AF1) domain, a DNA

binding domain, a short hinge region, and the C-terminal ligand binding domain

(LBD) where the activation function 2 (AF2) region is located, and where selective

ligand binding and allosteric protein interaction occurs. The general mode of action of

ER-α has been described as a tripartite model where the ligand, receptor, and specific

co-regulator complex is necessary to carry out target specific genomic modulation.15

In addition to its important homeostasis functions, ER-α plays a major role in

the progression of estrogen responsive breast cancer and several other diseases such

as osteoporosis, obesity and ovarian cancer. The importance of ER-α as target of

current breast cancer therapies and as a general model for the action of NRs has

attracted much attention since its isolation by Green et al. 13.

The action of SERMs can be explained by the presence of coregulator proteins

that direct the tissue specific action of ER. The general mode of action of ER-α has

been described as a tripartite model where the ligand, receptor and specific

coregulator complex is necessary to carry out target specific genomic modulation. In

the next section will describe the structure and function of ER-α and the effect of

ligand and coregulator proteins in determining its biological activity 15.

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Figure 1.2: Schematic representation of functional domains in full length ERα (A). Model of ERα-LBD showing ligand induced conformations of helix 12 for agonist (purple, 1ERE) and antagonist (red, 3ERT) ligands (B). Molecular models were generated with VMD using research collaboratory for structural bioinformatics protein data bank (RCSB-PDB) files 1ERE and 3ERT .16,17

1.3 Estrogen Receptor role in breast cancer Hormone molecules are essential for normal homeostasis of multicellular eukaryotic

organisms. In humans, hormones are involved in proper development and

homeostasis of virtually every tissue type. Estradiol (E2) is known as the female

AF-1 DNA-BD LBD AF-2

1 552302

His6-ERα-LBD (302-552)

A

B

16

hormone; however, it is present in males as well. In addition to its important effect

on development and in homeostasis, E2 has been directly linked to several disease

states such as breast cancer, colon cancer, ovarian cancer, osteoporosis and obesity.

Estrogenic signaling is mediated by the action of ERs. There exist two major

ERs, ER-α and ER-β. Although they share limited sequence similarity, X-ray crystal

structures show that they have similar conformations. ER-α constitutes the majority

of the estrogen receptors present in the cell and will be the focus of this thesis work.

ER-α plays a crucial role in the growth and progression of approximately two

thirds of diagnosed breast cancers, know as estrogen responsive breast cancer. As

early as the 1800s it was observed that oophorectomy (surgical removal of the

ovaries) caused tumor regression on a substantial portion of premenopausal women

affected by metastatic breast cancer. This pointed to an important role of estrogen in

tumor progression as the ovaries are the major estrogen producing tissue. In the

1950’s it was after Janson and Jacobson purified ER-α and showed that estrogen was

targeted to specific tissues that the molecular mechanism started to be elucidated 18.

Since cloning of the ER-α in the 1990’s a great deal has been learned about its

function and structure.

ER-α is a major target of breast cancer therapy because it has been

demonstrated that the majority of human breast cancers overexpress this receptor.

Compounds have been synthesized that bind to ER-α with high degree of selectivity

and antagonize the effect of estradiol, thus inhibiting the estrogenic response. One of

the classic examples of pure antiestrogens in the drug Fulvestrant (ICI182,780) which

binds the ER resulting in antagonistic effects and downregulation of the estrogen

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response 19. Selective estrogen receptor modulators (SERMs) such as Tamoxifen and

Raloxifene show competitive binding with ER and display agonist or antagonist

activity in a tissue specific manner 20.

Figure 1.3: Schematic view of ER-α activation by ligand binding and coregulator directed transcriptional activity. Ligand binding induces a conformational change that releases HSP90 and favors formation of ER dimers. Co-activator or co-repressor protein complexes can then interact with the AF-2 (see text) region and direct the transcriptional activity towards specific genomic targets.

HSP90

HSP90

ligand binding

dimerization

Co-A Co-R

18

1.4 Site directed spin labeling and EPR: application to protein dynamics and structure

(a) Measuring local dynamics Site directed spin labeling (SDSL) is an emerging technique that allows the

introduction of an EPR active nitroxide probe anywhere in the sequence of a protein

of interest 21. It consists of preparing a construct with one or more reactive cysteines

at the points of interest. Spin labels containing the EPR active nitroxide moiety and a

thiol specific reactive group are then used to quantitatively label the exposed cysteine,

thus producing stable nitroxide modified proteins that can be assayed by EPR.

Several nitroxide labels have been developed that covalently bind reactive thiols, two

of which are displayed in figure 1.4. In this work we have used a custom label that is

expected to represent the local environment more accurately.

O

SS

N ON

S

Figure 1.4: Molecular structures of cysteines derivatixed with two nitroxide spin labeles: methanethiosulfate spin label (MTSL) and iodomethyl spin label (IMSL).

SDSL has several advantages over methods that have been more commonly

applied to study protein dynamics, like X-ray crystallography, fluorescence labeling

and nuclear magnetic resonance (NMR). The most important advantage is the

MTSL IMSL

19

sensitivity of EPR to dynamics on the time scale of protein domain motion. This

means that EPR is applicable at physiologically relevant conditions and

concentrations, another distinct advantage of EPR over most other structural methods.

In addition, distances can be measured quite accurately in doubly labeled systems,

and the method does not suffer from the same difficulties with adventitious quenching

that are present with fluorescent probes.

These features make SDSL an ideal tool for probing dynamics and structure of

the ER and its molecular level response to potential drugs for breast cancer treatment.

Nitroxide spin labels are very sensitive reporters of local dynamics and structure. By

introducing a spin label at a point of interest it is possible to learn about local

structure by interpreting distinctive lineshape features of the EPR spectrum. Spin

labeled proteins yield spectra that to a first approximation can be explained by two

coexisting dynamic processes. Assuming the overall motion of the protein can be

frozen out by viscous solvents (for a globular 30 KDa protein a 10% glycerol buffer

will reduce the overall tumbling to the hundreds of ns time scale, thus the protein

rotation is essentially frozen in the EPR timescale) the remaining dynamics will be

due to internal rotameric freedom of the label within the local environment and

flexibility of the backbone. Although some theoretical models have been described to

account for these two factors, it still remains a challenge of EPR analysis to

distinguish the two types of dynamics reliably 22,23.

In this work we have used a nitroxide spin label with shorter tether connecting

it to the reactive thiol. In principle, this will reduce the internal degrees of freedom

and make our spectra more representative of the local backbone dynamics. Rotation

20

around the χ1, χ2 and χ3 bonds are unlikely due to high energetic barriers and steric

constraints (see Figure 1.5). It has been found that motions relevant to EPR relaxation

(in the nanosecond time scale) can be described by rotations around χ4 and χ5 of the

standard MTSL probe. The “χ4, χ5” model is the most accredited by experimental and

MD studies 24,25. A considerable amount of effort has been put into reconciling EPR

spectra with MD calculations as a means to both improve the fitting of dynamic

parameters to experimental spectra and to fine tune the physical interpretation of

those parameter to derive structural information. Most studies take as a standard the

nitroxide label methanethiosulfonate spin label MTSL, which is the most common

protein spin label . Some important differences are immediately apparent between

MTSL and the IMSL probe used in this study: (i) when bound to a protein IMSL has

a 1 carbon shorter tether to the backbone (ii) IMSL is connected to the reactive Cys

though a thioether linkage which is resistant to reductive cleavage. These differences

are expected to have important consequences to the dynamics of the label, in

particular the removal of a torsional angle should be considered when interpreting the

results.

A simple semi-quantitative approach can be used to describe local dynamics.

The inverse width of the central resonance line (ΔH0−1 ) can be used to as a measure

of nitroxide mobility that accounts for effects of ordering as well as correlation time

of the motion 26-28.

21

Figure 1.5: Cysteine specific reaction of IMSL with an exposed thiol (i). Molecular dynamics results of showing the rotameric state for the χ2, χ3, χ4 torsion angles.

χ2 χ3 χ4

(i)

(ii)

χ1

χ2

χ3

χ4

22

(b) Measuring distance by EPR EPR is a versatile technique that in many ways is ideally suited to study

complex macromolecules. The previous section described the utility of EPR to study

local environment by SDSL. The same approach can be used to extract accurate

distance constraints between two unpaired electrons. The coupling between two

electrons is dominated by the spin exchange interaction for distances less than 10 Å,

and by the magnetic dipolar interaction for distances between 10 Å and 70 Å 29,30.

Most useful for determining interspin distances is measurement of the dipolar

interaction which has an inverse cubic dependence on the distance separating the two

radical centers. In this thesis we describe the use of two EPR techniques to measure

distances: continuous wave EPR (CW-EPR) and pulsed double electron-electron

resonance (DEER) spectroscopy.

The two methods are technically different but they measure the same

underlying physical parameter, dipolar coupling. This can be thought of as the

interaction occurring between two magnetic dipoles and it is the same effect that

gives rise to nuclear Overhauser effects (NOE) in NMR. In essence, the magnitude of

this interaction is inversely proportional the cube of the distance separating the two

centers. For short distances (5 – 20 Å) this interaction can be observed directly in the

CW-EPR spectra of frozen samples. In this range the dipolar coupling interaction

results in a broadening of the resonance lines that is mostly observable in frozen

solution because the effect tends to be averaged out in fast tumbling liquid samples.

By deconvoluting the spectrum of a sample with interacting labels from a spectrum

with single (non-interacting) labels one obtains a Gaussian broadening function

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whose width reflects the dipolar coupling and therefore the separation between the

labels. For distances above 20 Å a more sophisticated pulsed method needs to be used

to evaluate the dipolar interaction 31.

DEER is an EPR technique that uses microwave (mw) pulses to measure

coherent interactions between two spins located within a 70 Å radius. Coupling

between the localized electrons on the radicals can be used to determine distances on

a scale that is difficult to access by other methods. Recently, technical advances in

instrument design have taken this technique to the forefront of structural biology.

Important insights into protein structure have been gained by applying SDSL and

DEER spectroscopy to systems which were intractable by more conventional means

such as X-ray crystallography or NMR.

1.5 Spectroscopy

(a) CW-EPR Electron Paramagnetic Resonance (EPR) is a magnetic resonance technique sensitive

to the presence of unpaired electrons. In general, when an electron is placed in an

external magnetic field (B) the spin degeneracy is lifted, giving rise to two energy

levels. This effect, known as the Zeeman interaction, arises from the fact that the

electron possesses both a charge and a magnetic moment (µB). The magnetic moment

is quantized along the external field direction, and may have a projection along this

axis that is either parallel or antiparallel with respect to the applied field, leading to an

energy difference between the two states. According to Boltzman’s equation, the low

energy state will have higher population than the higher energy state, making it

24

possible to observe absorption of radiation at the resonance frequencies. These

concepts are summarized pictorially in Figure 1.6.

Figure 1.6: The magnetic moment (μ) of an electron depicted as arising from the rotating charge (i). An energy diagram representing the effect of an applied magnetic field on the energy states of an unpaired electron (ii). First order energy Hamiltonian summarizing the major interactions affecting the EPR spectrum: the Zeeman interaction and the hyperfine interaction apply to all cases where an electron is coupled to a nucleus while the exchange interaction and the dipolar interaction become relevant for the case of two interacting spins (iii).

In nitroxides the unpaired electron is associated with a nitrogen nucleus giving

rise to a hyperfine interaction. 14N has a nuclear moment of 1 and 99.6% natural

Ener

gy

Field

νh

2121 SDSSJSSAISgBH B ⋅⋅+⋅+⋅⋅+⋅⋅= μ

Zeeman interaction

Nuclear Hyperfine interaction

Exchange interaction

Dipolar interaction

(i)

(iii)

(ii)

µ = g β S

Magnetic moment Zeeman splitting

Spin Hamiltonian

25

isotopic abundance. The (2I+1) energy levels, +1, 0, −1, correspond to the allowed

states on the 14N nucleus. Therefore the nitroxide spectrum in the fast motional

regime is composed of a triplet centered at the giso=2.004 resonant field.

(b) Effect of molecular motion on EPR spectrum of nitroxides in solution Fast limit: The g value of a bound electron generally deviates from the ge of an

isolated electron due to spin-orbit coupling centered on the atoms with significant

unpaired electron spin density. The coupling increases strongly with increasing

atomic number of such nuclei.. As the orbital angular momentum is fixed in the

molecular frame and the spin becomes quantized along the direction of the applied

field, the g-value depends on the orientation of the molecule with respect to the field.

This anisotropy can be described by a tensor with three principal values, gx, gy, and

gz.

In fluid solutions the molecular tumbling rates are much higher than the

differences of the electron Zeeman frequencies between different orientations. In this

situation the g value is orientationally averaged and only the average value can be

measured giso = (gx + gy + gz )/3.

The interaction of the unpaired electron with nuclear spins splits each energy

level into sublevels. As in the case of the g tensor, the hyperfine interaction also

displays anisotropy. Each hyperfine tensor is characterized by three principal values

Ax, Ay, Az, and by the three angles that specify the orientation of its principal axes

system relative to the molecular frame defined by the g tensor.

26

Fluid systems with fast molecular tumbling cause motional averaging of the

hyperfine observable resulting in Aiso = (Ax + Ay + Az)/3. This is due to the Fermi

contact interactions of electrons that reside in an s orbital of the nucleus under

consideration. Anisotropic contributions to the A tensor result from spin density in p,

d, or f orbitals on the nucleus. For a nitroxide, the nuclear spin I = 1 on the 14N atom

is the major nucleus coupled to the electron spin S = 1/2 that resides in the pz orbitals

on the N and O atoms. The hyperfine coupling causes a splitting of the electron spin

levels into three sublevels. As the field is swept with constant microwave irradiation,

three resonance peaks are observed. The nuclear Zeeman interaction shifts the mI =

+1 and mI = −1 sublevels to lower and higher energy respectively. Figure 2.8 shows a

typical spectrum of a nitroxide in solution displaying g and A averaging. EPR spectra

are acquired by field modulation and as a result the resulting signal is usually

displayed in derivative mode.

Rigid limit: We have discussed the effect of an unpaired electron with the nuclear

spin coupled to the 14N atom as in the case of a nitroxide radical. The EPR spectrum

is dominated by the hyperfine interaction of the electron spin with the nuclear spin of

14N and the g value is shifted due to anisotropic spin-orbit coupling between the lone

electron and the 2pz orbital where the spin is located. The isotropic contribution to the

hyperfine interaction observed in fast tumbling cases is due to Fermi contact

interaction in the 2s orbital of the nitrogen. The anisotropic contribution comes from

the spin density on the nitrogen 2pz orbital, the hyperfine interaction is large in the Az

direction and smaller in the Ax and Ay direction due to the orientational dependence of

the electron-nuclear dipolar interaction. Conversely, g shifts are also dependent on the

27

orientation of the 2pz orbital with respect to the external magnetic field. For instance,

in a nitroxide gx represents the case when the field is along the N-O bond. Therefore

if we consider a frozen sample (or powder) where all the orientations are equally

probable, we expect that the triplets of lines at different orientations of the nitroxide

in the applied field will have different splitting, as well as being shifted with respect

to each other. The sum of all the contributions at different orientations gives rise to a

characteristic “powder pattern” displayed in Figure 1.7.

Slow motion regime: The mobility of a spin probe depends on the local viscosity and

its connectivity to a larger macromolecule (e.g. proteins, nucleic acids, polymers). In

the case of a spin label, the mobility depends on the flexibility of the tether

connecting it to the macromolecular backbone, the dynamics of the backbone itself,

and on the global tumbling of the protein as a whole. A measure of the mobility is the

rotational correlation time τr, which corresponds to the characteristic time during

which a molecule maintains its spatial correlation. If the inverse of τr is on the same

order of magnitude as the anisotropy of an interaction, this interaction is partially

averaged and the EPR spectrum will become sensitive to τr and the specific dynamics

experienced by the probe. For nitroxides at X-band the EPR spectrum is dominated

by the hyperfine anisotropy of 150 MHz, which makes the spectra sensitive to

correlation times of the order of a few nanoseconds. Figure 2.8 displays a spectrum in

the intermediate motion regime.

28

Figure 1.7: Representative EPR spectra of nitroxides exhibiting the three dynamic cases described in the text. From the top: the fast motional case, the intermediate case and the rigid limit case.

Τc = 4 x 107 s-1

3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450-8

-6

-4

-2

0

2

4

6

8x 10

-3

3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450-3

-2

-1

0

1

2

3x 10

-3

Τc = 1 x 109 s-1

Τc = 1 x 106 s-1

Fast Limit

Rigid Limit

29

(c) DEER spectroscopy Double electron-electron resonance (DEER) spectroscopy is a pulsed EPR

technique that makes it possible to measure inter-spin distances larger that 2 nm with

high precision. Distance measurement by EPR is based on dipole-dipole couplings

between the unpaired electrons found on nitroxide radicals. In addition to dipole-

dipole interaction a short range exchange interaction is also present between

uncoupled electrons, but can be neglected at separations greater than 1 nm. In this

case a good approximation is to assume that the dipolar coupling acts between two

point dipoles.

The 3+1-pulse DEER sequence displayed in Figure 1.8 consists of a two-pulse

echo sequence π/2-τ-π on a fixed observe microwave frequency (ω1) and an

additional microwave pulse on a fixed pump frequency (ω2) whose temporal position

is varied between the positions of the two observer pulses. The principle can be

understood in terms of two coupled spins A and B. The A spins are influenced only

by the observer sequence while the B spins are influenced only by the pump pulse

and do not contribute directly to the signal. The π/2-τ1- \π sequence at frequency ω1

acts on spin A to induce a spin echo at τ1. During the evolution of this echo, ω2 is

pulsed at time t to produce a π/2 rotation of the B spins. An evolution time τ2 is

chosen based on relaxation parameters, and is followed by a third pulse at ω1 that

refocuses the remaining spin polarization on the A spins and results in a spin echo at

time τ2 after the pulse. The DEER signal is acquired by measuring the area under the

refocused echo signal and plotting it versus t.

30

Figure 1.8: (i) Schematic diagram displaying the pulse sequence employed in DEER spectroscopy. (ii) Model of a system of coupled spins where the inter-spin distance (dashed line) represents the measurable parameter up to 70 Å

The specific DEER experiments performed here all deal with measuring interspin

distance between nitroxide centers. In this case selection of spins A and B is achieved

by orientational selectivity. Figure 1.9 shows an EPR spectrum of a frozen nitroxide

with indicated the positions of the pump and probe frequencies. By selecting these

positions we are effectively irradiating different populations of the spin probe: the

observe frequency will preferentially excite nitroxides with the Z magnetic axis

parallel to the external field, while the pump frequency will irradiate nitroxides with

the X and Y magnetic axes aligned to the field.

τ1 τ1 τ2 τ2

t

π/2 π π

π

ω1

ω2

Pulse sequence

B0

ON O

N

A B

(i)

(ii)

31

Figure 1.9: Non-derivative absorbance spectrum of a nitroxide showing the position of the observe (ω1) and pump (ω2) frequencies chosen to carry out DEER experiments.

Analysis of the DEER spectrum resulting from a set of coupled spins entails first the

subtraction of a concentration dependent background signal, then the deconvolution

of the time domain signal (Eq 1.2) to the frequency domain (Eq 1.3) from which the

dipolar coupling can be measured (Eq 1.4).

Frequency

ω1

ω2

32

[Eq. 1.2]

]sin))cos(1(1)[exp()(2/

0

θθωλπ

dttkCFtV eeBDEER ∫ −−−=

[Eq. 1.3]

Jddee +−= )1cos3( 2θωω

[Eq. 1.4]

3

2 12 r

gg BohrBAdd

μω =

[Eq 1.5] 3

2 12 r

gg BohrBAdd

μω =

Figure 1.10: Summary of the analytical equations used to derive interspin distances from DEER spectra. The figures represent experimental and calculated fits derived from the corresponding equations where C is the concentration of spin probe, λ is the modulation depth, FB is the fraction of B spins excited by the pump pulse, ωee is the electro-electron coupling and k is a constant.

1.6 Overview The flexibility inherent within all protein structures has long been recognized as an

essential characteristic of receptor-ligand interaction. A careful consideration of the

traditional “lock and key” mechanism of ligand-receptor interaction reveals

inconsistencies when presented with situations where the ligand binding pocket is

located within the protein. In this case it is clear that the conformational changes must

occur that allow ligands to access the binding site. Although crystal structures

produce precise snapshots of single configurations they often are unable to provide

any information about conformational flexibility. Furthermore, protein crystals are

33

grown under non-physiological conditions and therefore the structures derived from

them might not always represent a physiologically relevant conformation.

Few experimental techniques are sensitive to the conformational dynamics

taking place within proteins. NMR spectroscopy can be used to derive structural

constraints for small (< 20 kDa) proteins in solutions, however it suffers from

important limitations such as very low sensitivity that requires proteins to be in mM

concentrations and low resolution, particularly for highly flexible structures. These

limitations are not present in SDSL-EPR. As discussed earlier, this technique can be

used to specifically measure ns timescale dynamics, and extract accurate distance and

distance distribution constraints that reflect conformational flexibility. In this thesis

we apply the emerging technique of SDSL and EPR to study protein flexibility and

the effects of ligand, allosteric and other environmental parameters such as

hydrophobic mismatch and electrostatics on conformational flexibility of proteins.

In Chapter 3, we investigate the dynamic response of the human estrogen

receptor α ligand binding domain (ERα-LBD) to the binding of different ligands and

coactivator peptides SDSL-EPR and DEER spectroscopy. We describe how local

changes in dynamics at position 543 of the C-terminal helix 12 (H12) domain has

provided the first direct experimental demonstration that this domain undergoes

dynamic changes in response to ligand binding that correlate with the ligand’s

biological activity. In line with the driving theme of this work, the local dynamic

results obtained are incorporated into the already existing crystal structure model

thus, completing the static picture available by the addition of protein flexibility

measurements.

34

Ligand-dependent changes are also observable for a label positioned at residue

530 in the H12 hinge region, however much more dramatic changes are observed at

this position in the presence of both ligand and coregulator peptides. We investigated

the ligand/coregulator induced changes in structure by measuring interspin distances

in 530 labeled ERα-LBD dimers using DEER. These results extend the current model

of ERα-LBD action and provide dynamic information on the H12 region as well as

quantitative structural information on the dimer conformation of ERα-LBD in

solution. The results suggest a large-scale remodeling of the ER dimer complex that is

sensitive to details of the ligand structure as well as the nature of the coactivator

sequence.

In Chapter 4 we continue our investigation of the receptor’s flexibility by

looking directly at the interaction between ligand and receptor using two nitroxide

labeled estradiols, HO-2105 and HO-2447. These new compounds proved to be

excellent molecular probes of ligand/receptor interaction with ERα-LBD. EPR

spectroscopy was used to investigate binding properties and local dynamics of the

spin-labeled ligand. Fluorescence spectroscopy demonstrated quenching of both the

fluorescence of estradiol and on the intrinsic tryptophan fluorescence of ERα-LBD by

the nitroxide moiety of the labeled ligand. We describe two methods to assay binding

of the probes to ERα-LBD: (i) an EPR derived binding assay and (ii) an intrinsic

tryptophan fluorescence quenching binding assay. Saturation binding studies of the

estrogen probes using the two assays showed good agreement between the

independent techniques. DEER spectroscopy was used to measure interspin distances

35

between the bound probes in the ERα-LBD homodimer complex. The structural

results are consistent with X-ray crystals of the ERα-LBD dimer and provide new

information about the distribution of conformations in the homodimer. The spin

labeled estradiols here described serve as versatile probes of ligand binding, local

dynamics and structure with potential applications as ER selective imaging agents

and as oxidative stress probes.

Chapter 5 includes results obtained on projects not related to ER, but all

sharing the broader theme of application of EPR to study biophysical systems where

we aim to gain insight into protein flexibility. EPR is a versatile technique that

enables study of a variety of biophysical phenomena. Here we describe applications

of EPR as a method to evaluate singlet oxygen production (1O2), characterize

conformational effects of hydrophobic mismatch on transmembrane helices, develop

methods for characterizing protein self-assembly and characterize unstructured

protein domains. Furthermore, as these projects result from collaborations with

research groups from different disciplines such as organic chemistry, medicinal

chemistry, biochemistry and mechanical engineering they each add a particular set of

challenges.

Chapter 6 evaluates future directions for continuing the investigation into the

molecular basis of ER action. Here we develop a theoretical model for the effect of

ligand/coregulator interaction on ER that is based on the combined results previously

presented. We also consider foreseeable challenges and provide recommendations

relative to experimental design on future investigations of ER with SDSL.

36

Summary Chapter 1 introduces background information on the nuclear receptor (NR)

superfamily. The relevance of estrogen receptor (ER), a member of the NR

superfamily, is discussed as well as an overview of its structure and function. This

Chapter also introduces site directed spin labeling (SDSL) and electron paramagnetic

resonance (EPR) as they apply to the study of protein dynamics and structure. Here

we provide the theoretical background necessary to interpret the experimental results

presented in later chapters. The study design and experimental procedure used to

produce and characterize the necessary ER mutants is described in Chapter 2. In this

chapter we also address so particular concern regarding method development that can

be helpful when optimizing recombinant protein production.

37

Chapter 2: Methods

38

2.1 Study Design The previous chapter establishes the relevance of ER-α as an important target

for breast cancer research and treatment. Antagonist ligands tend to inhibit cancer

growth; therefore it is desirable to understand the specific effect of antagonists and

SERMs on the structure of the receptor as a way to design more potent and more

selective inhibitors of the carcinogenic pathway. It is clear that the complex

pharmacology of estrogens cannot be fully explained by the static conformations of

ER-LBD available from X-ray crystallography. Dynamics of the LBD in response to

different ligands has been implicated in the response of receptors to SERMs 32. The

study described here is designed to combine new physical methods and synthetic

strategies to answer questions about the dynamic behavior and structure of ER that

cannot be addressed comparably by other means.

The ligand series selected for this study have similar binding modes, but

different biological responses ranging from full agonist to partial antagonist to full

antagonist 33-36. Their common estradiol steroid scaffold (Fig 2.1), orients the ligand

in the binding pocket, conserving the interaction of hydroxyl groups at position 3 with

Glu353, Arg394 and at position 17 with His524 respectively 37.The different activities

of the compounds can be attributed to the presence of sidechains at the 11β or 7β

positions as in the case of the antagonist ICI-182-780. Figure 2.1 shows the effect of

11β alkyl substituents on the biological activity of estradiol derivatives as described

by Hochberg et al 34,36.

39

Figure 2.1: Estradiol scaffold showing the 11β position and the effect of alkyl chains of variable length.

Increasing the number of carbons at the 11β position effectively tunes the biological

response of the ligands. By exploiting this recent finding it is possible to

systematically study the effect of agonist SERMs and antagonist on the dynamics and

structure of the H12 region.

Ligand binding is the first step in the estrogenic pathway; however,

coactivator binding determines the final transactivation function of ER. For this

reason it is important to include the effect of allosteric interaction via the AF2 region

as part of a comprehensive characterization of the structural changes occurring during

ER’s activation. We used co-activator derived peptides (D22, SRC-1, TIF-2, RIP-

140), containing the conserved LXXLL motif to investigate the effect of allosteric

interaction. These peptides are known to bind in a ligand dependent fashion to ER-α,

and are commonly used as predictors of a ligands’ biological activity 38.

2.2 Specific aims The following specific aims were identified in the planning stages of this research:

I. Produce mutants of ERα that will allow attachment of spin labels at

strategic points on helix 12

OH

OHCn, 11β n = 0 – 2; agonist n = 3 – 5; SERM n > 5 or bulky; antagonist

40

II. Measure the specific structural and dynamic differences that accompany

binding of agonists and antagonists

III. Measure the specific structural and dynamic differences that accompany

binding of ligand dependent coregulator peptides

IV. Characterize a new class of spin labeled estradiols for applications as

probes of ligand/receptor interaction

The long term objective of this project is to develop a rational basis for designing

therapeutic agents for hormone dependent breast cancer by elucidating the detailed

structural and dynamic changes of the receptor in response to agonist and antagonist

ligands

2.3 Site directed mutation

(a) Selection of mutation sites and site directed mutagenesis Mutations of ER for SDSL have been selected with careful attention to avoiding

possible perturbations of the interactions of H12 with the ER and other proteins. A

number of amino acids that critically mediate the role of H12 in ER’s action have

been identified by various studies 39,40. In the agonist conformation of ER-α

complexed with estradiol, H12 is positioned over the binding pocket. In this

configuration, the underside of H12 (specifically residues L540, L544, and A546)

undergoes a hydrophobic interaction with the receptor, while the outer surface forms

a charged binding region for coactivator docking (AF2). Mutation studies have shown

that the negatively charged amino acids D538, E542 and D545 are necessary for AF2

interaction and subsequent transcriptional activity. From this information it was

deduced that position M543 provides the most likely candidate for SDSL. Molecular

41

modeling on this site indicates that it is most likely to reflect significant ligand

induced changes while minimizing the perturbations to the critical interactions of

H12. Other possible sites meeting these criteria include mutations on L539 and L541.

Based in this information the sites for SDSL were selected to be 543 and 530. Herein

we describe the production of four ER-LBD mutants in addition to the wild type

variant.

Table 2.1: The sequences of the DNA primers used for site directed mutagenesis Primer name Sequence 5’-3’ C381S CCACCTTCTAGAAAGTGCCTGGCTAGAG C417S GAACCAGGGAAAAAGTGTAGAGGGCATG C530S GTACAGCATGAAGAGCAAGAACGTGGTGCM543C(1) CTGCTGGTGGAGTTGCTGGACGCCC M543C(2) CTGCTGGTGGAGTGGCTGGACGCCC M543C(3) CTGCTGGTGGAGTGCCTGGACGCCC

(b) Expression, purification and spin labeling A pET15b vector was used to clone and express ERα-LBD (a.a. 302-552).

This vector encodes for a histidines tag region and thrombin cleavage site at the N-

terminus of the expressed protein. This feature allows for convenient purification with

a Ni-NTA column.

Three mutant constructs plus the wild type ERα-LBD were prepared for the

purpose of this study. The initial step for the production of mutants is the design of

primers to encode the specific mutation. Table 2.1 summarizes the primers used to

achieve the necessary mutations.

The mutants produced are derived from the wild-type ERα ligand-binding

domain (hERα-LBD) (a.a. 302-552) expressed in a pET15b vector 41. The wild type

42

protein contains four cysteines, three of which (C381, C417, C530) are solvent

exposed and therefore reactive to electrophilic labeling, the fourth (C447) is buried

and proven to be unreactive to labeling 42. The mutant construct C318S/C417S was

used to allow the selective labeling of C530. Production of a singly reactive cysteine

at position C543 was accomplished by mutagenesis of M543C in the cysteine-less

construct.

Plasmid mutation was carried out using the QuikChange Kit from Stratagene,

Inc (San Diego, CA). Purification of the plasmids was accomplished with Qiaprep

spin columns (Qiagen) and their sequences confirmed by SeqWright (Houston, TX)

after every round of mutation. Plasmid amplification was achieved in XLt-Blue E.

coli cells (Novagen, San Diego, CA), while protein production was carried out in Bl-

21(DE3)pLysS E. coli (Novagen) in low salt LB media, 18 hour incubation at 37 °C,

followed by induction with 0.5 µM IPTG at 37 °C for 3 hours. Cell pellets were lysed

and solubilized using non-denaturing conditions (2 M NDSB, 10 mM Tris, 100 mM

KCl, 0.2 mg/ml egg white lysozyme from Sigma, St. Louis, MO) and sonication at

4 oC to reduce viscosity.

His tagged ER was purified from the cell lysate with a Ni-NTA affinity column

by a flow through method using standard elution in 300 mM imidazole. Labeling was

accomplished by incubating the eluate, which typically had protein concentration

between 10 and 30 mg/ml, with 5 fold molar excess of iodomethyl spin label (IMSL,

see below) for a minimum of 4 hours. Fractions containing the protein were then

assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),

43

and pooled. A ten fold dilution of the pooled fraction with 50 mM Tris, 10% glycerol

at pH 8.1 was used for loading onto a pre-equilibrated cation exchange column. After

extensive washing to remove unreacted label, the elution was carried out with 50 mM

Tris, 10% glycerol at pH 8.1 buffer containing 200 mM KCl. Samples prepared by

this method were found to be >90% pure when assayed by LC-MS. If necessary, the

sample was further purified with Zeba desalting spin columns (Thermo Scientific,

Rockford, IL) to remove residual spin label.

(c) Characterization of purified mutants by LC-MS and [3H]estradiol binding assay Characterization of the mutants and wild type constructs was carried out by

independent measurements. Plasmid sequence was verified by SeqWright (Houston,

TX), MS analysis of the purified protein was conducted by Novatia (NJ) and

[3H]estradiol binding was carried out by the group of Prof. John Katzenellenbogen at

the University of Illinois at Urbana-Champaign (IL). The results of these analyses are

summarized in the following figures.

44

ERα-LBD wild type ATGAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 60 GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 120 TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 180 GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 240 TGTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGGG 300 AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATG TGTAGAGGGC 360 ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 420 CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 480 TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 540 AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 600 CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCAGGCACAT GAGTAACAAA 660 GGCATGGAGC ATCTGTACAG CATGAAGTGC AAGAACGTGG TGCCCCTCTA TGACCTGCTG 720 CTGGAGATGC TGGACGCCCA CCGCCTACAT GCGCCC MKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINW 60 AKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEG 120 MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD 180 KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLL 240 LEMLDAHRLHAP 252 N-terminal tag = GSSHHHHHHSSGLVPRGSH LC-MS Analysis: 30824 Da target mass

Figure 2.1: cDNA sequencing results with the derived amino acid sequence (A). LC-MS results confirming the target molecular weight for the ER mutant (B)

A

B

45

ERα-LBD cysteine free ATGAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 60 GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 120 TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 180 GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 240 TCTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGGG 300 AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATC TGTAGAGGGC 360 ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 420 CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 480 TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 540 AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 600 CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCAGGCACAT GAGTAACAAA 660 GGCATGGAGC ATCTGTACAG CATGAAGTCC AAGAACGTGG TGCCCCTCTA TGACCTGCTG 720 CTGGAGATGC TGGACGCCCA CCGCCTACAT GCGCCCTGAG GATCCGGCTG CTAACAAAGC 780 CCGAAAGGAA NCTNN Peptide Sequence: MKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINW 60 AKRVPGFVDLTLHDQVHLLESAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKSVEG 120 MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD 180 KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKSKNVVPLYDLL 240 LEMLDAHRLHAP 252 N-terminal tag = GSSHHHHHHSSGLVPRGSH Target mass = 30776 Da

Figure 2.2: : cDNA sequencing results with the derived amino acid sequence (A). LC-MS results confirming the target molecular weight for the ER mutant (B).

A

B

46

ERα-LBD C543 (C381S, C417S, C530S, M543C) ATGAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 60 GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 120 TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 180 GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 240 TCTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGGG 300 AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATC TGTAGAGGGC 360 ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 420 CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 480 TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 540 AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 600 CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCANGCACAT GAGTAACAAA 660 GGCATGGAGC ATCTGTACAG CATGAAGTCC AAGAACGTGG TGCCCCTCTA TGANCTGCTG 720 CTGGAGTGTC TGGACGCCCA CCGCCTACAT GCGCCCTGAG GATCCGGCTG CTAACAAAGC 780 CCGAAAGGAA GCTGANTTN Peptide Sequence: MKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINW 60 AKRVPGFVDLTLHDQVHLLESAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKSVEG 120 MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD 180 KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIXHMSNKGMEHLYSMKSKNVVPLYXLL 240 LECLDAHRLHAP 252 N-terminal tag = GSSHHHHHHSSGLVPRGSH

Figure 2.3: cDNA sequencing results with the derived amino acid sequence (A). LC-MS results confirming the target molecular weight for the ER mutant (B).

A

B

47

ER-M5 C543-C530 (C381S, C417S, M543C) ATGAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 60 GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 120 TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 180 GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 240 TCTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGGG 300 AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATC TGTAGAGGGC 360 ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 420 CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 480 TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 540 AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 600 CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCAGGCACAT GAGTAACAAA 660 GGCATGGAGC ATCTGTACAG CATGAAGTGC AAGAACGTGG TGNCCCTCTA TGACCTGCTG 720 CTGGAGTGTC TGGACGCCCA CCGCCTACAT GCGCCCTGAG GATCCGGCTG CTNACAAAGC 780 CCGAAAGGAA GCTGAGTNN MKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINW 60 AKRVPGFVDLTLHDQVHLLESAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKSVEG 120 MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD 180 KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVXLYDLL 240 LECLDAHRLHAP 252 N-terminal tag = GSSHHHHHHSSGLVPRGSH Target Mass = 30764 Da

Figure 2.4: cDNA sequencing results with the derived amino acid sequence (A). LC-MS results confirming the target molecular weight for the ER mutant (B).

A

B

48

ERα-LBD C530 (C381S, C417S) ATGAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 60 GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 120 TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 180 GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 240 TCTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGGG 300 AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATC TGTAGAGGGC 360 ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 420 CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 480 TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 540 AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 600 CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCAGGCACAT GAGTAACAAA 660 GGCATGGAGC ATCTGTACAG CATGAAGTGC AAGAACGTGG TGCCCCTCTA TGACCTGCTG 720 CTGGAGATGC TGGACGCCCA CCGCCTACAT GCGCCCTGAG GN MKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINW 60 AKRVPGFVDLTLHDQVHLLESAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKSVEG 120 MVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLD 180 KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLL 240 LEMLDAHRLHAP 252 N-terminal tag = GSSHHHHHHSSGLVPRGSH LC-MS Analysis: 30792 Da target mass

Figure 2.5: cDNA sequencing results with the derived amino acid sequence (A). LC-MS results confirming the target molecular weight for the ER mutant (B).

A

B

49

Figure 2.6: [3H]binding assay performed by Kathryn Carlson, in the laboratory of Prof. J. Katzenellenbogen (University of Illinois, Urban-Champaign, IL). From the top: (i) ER-LBD wild type displaying a Kd of 0.15 nM consistent with reported values, (ii) spin labeled ER-LBD at position 543, displaying a Kd of 1.2 nM, (iii) spin labeled ER-LBD at position 530 displaying a Kd of 0.9 nM.

0. 2. 5. 7. 10. 12.0.0

0.0

0.1

0.1

0.20 0.2

0.3

0.3

nM

0 1 2 3 4 5 6 7 8 9 100.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055

Free (nM)

0.0 2.5 5.0 7.5 10.00.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55

nM

ER-543 Kd = 1.2 nM

ER-530 Kd = 0.85 nM

ERα-LBD wild type Kd = 0.15 nM

(i)

(ii)

(iii)

50

The results summarized above show that the ER mutants proposed have been

successfully mutated, expressed and purified. Due to reasons discussed in section 6.5,

“Challenges”, only the ERα-LBD wild type, the ER-543 and the ER-530 mutants were

assayed for binding activity by [3H]estradiol assay. The binding assay indicates that high

affinity is retained after mutation and spin labeling of the mutant constructs tested.

2.4 Pitfalls and optimization of experimental conditions Plasmid design: In order for SDSL to be applicable, reactive cysteines need to be added

or removed, in order to produce mutants with cysteines placed at the desired positions. In

the case of ER, of the four cysteines present in the sequence, three are solvent exposed

while a third at position 381 is buried and therefore non-reactive. The mutagenic primers

have been designed individually to incorporate the necessary point mutation. Some

important considerations that go into primer design are:

(i) Primers should be designed with similar annealing efficiency

(ii) Primers should be between 25 and 45 bases in length with a melting

temperature (TM) greater than 75oC.

(iii) The mutation should be close to the middle of the primer in order to

ensure maximum annealing efficiency of the extending ends of the primer.

(iv) Primers should be designed with a GC content of about 40% and terminate

with one or more C or G at the 3’ –end.

Some useful online tools are available to help design optimum primers for the desired

mutations. In this work all primers were checked with PrimerX online primer design tool

(“http://www.bioinformatics.org/primerx/index.htm”).

51

The QuikChange kit from Stratagene (La Jolla, CA) used to introduce and

amplify the point mutations includes all the necessary enzymes for the PCR and

transformation reaction. We used a thermo-cycler to perform PCR. Some optimization of

the PCR procedure was necessary to satisfactory amplification, in particular “extension

time” was found to have the largest effect on synthesis. The final conditions used for the

3.5 Kb plasmid are as follows:

Segment 1 – 1 min at 95oC – meting step

Segment 2 – 1 min at 95oC – melting step

Segment 2 – 1 min at 55oC – annealing step

Segment 2 – 9 min at 65oC – extension step

Segment 2 is repeated 30 times.

Transformation of the PCR products was accomplished using standard protocol

described in detain in the QuikChange instruction manual with a minor modification

regarding the concentrations of cells plated. We found that using 5 μL of the

transformation reaction mixture yielded an optimal amount of cells for selection. The

plating media was composed of 10 g/l LB-broth dry powder, 1 g/l Agar and brought up to

volume with deionized water. Ampicillin (50 μg/l final concentration) was added after

sterilization by autoclave (121oC, 40 min).

Media composition optimization: Several media compositions were screened in order to

optimize expression and recovery of ER protein. Some media tested were: (i) LB broth

(Sigma, St. Louis), (ii) Terrific Broth (Sigma, St. Louis) and (iii) combinations of

52

tryptone and yeast extract. We found that satisfactory condition for cell growth and

protein production are achieved with the following media composition:

16 g/L Tryptone

3.3 g/L Yeast extract

5 g/L NaCl

~ 5 ml/L of 1 M NaOH to adjust the pH to pH 7.2.

Induction conditions: Varying the time and temperature of induction is a common method

to optimize protein production in prokaryotic systems. In the case of ER we found that

induction with isopropylthiolgalactose (IPTG) when the cell concentration reached OD600

~ 2 substantially increased protein yield. The fermentation was inoculated in the media

described above and incubated in a shaking incubator for 15-18 hours at 37oC. When the

cell concentration reached OD600 = 2, the flasks were spiked with 150 μM IPTG and the

induction was allowed to continue at 37oC for 4 hours. The cells were then harvested by

centrifugation at 15000 RPM and stored at −20oC until needed for purification.

Extraction conditions: The extraction step is crucial to ensure proper protein folding.

Prokaryotic expression systems tend to produce inclusion bodies made of misfolded and

aggregated protein that is difficult to refold into a native state. We found that using

extraction buffer with high concentrations of urea or guanidine would yield large

amounts of protein but with very low affinity. In fact, these preparations would quickly

form large precipitates due to protein aggregation. We improved the process by including

Dimethylethyl-(3-Sulfopropyl)-ammonium (NDSB) in the extraction buffer, so that the

final composition of the extraction buffer used in this work is:

53

50 mM Tris, pH 8.1

150 mM KCl

10 % glycerol

2 M NDSB

1 mM PMSF

This extraction buffer produced active protein, although smaller total yields were

observed compared to denaturating buffer conditions.

Purification procedure optimization: The extracted protein was purified in a two column

process that reliably yielded more than 90% pure protein as assayed by SDS-PAGE and

LC-MS. The process consists of an initial capture step with Ni-NTA and a second

refinement step with a cation affinity resin. The purification protocol was described

earlier in this Chapter, however caution should be taken to reduce exposure of the protein

to room temperature. Care should also be taken to minimize the time that the protein

spends in the Ni-NTA elution buffer due to the presence of imidazole. Under optimal

conditions the two chromatographic separations should be carried out in the same day.

DEER acquisition: DEER experiments can accurately provide two important parameters

about interacting spins: (i) distance and (ii) distance distribution. These two parameters

affect the DEER signal in different respects: the distance information is encoded in the

first μs of signal evolution, while the distance distribution might require acquisition up to

several μs in order to be fully resolved. The analysis method used allows us to estimate

the uncertainty for the two parameters. We have developed a method of “baseline

stitching” to address cases where the distance distribution information cannot be easily

54

separated from the baseline. In this method we take spectra at a short evolution time and

spectra at long evolution time and use the initial part of the spectrum to derive distance

constraints and the latter part to properly subtract the baseline. This method holds

promise for cases such as particularly flexible proteins where the distance distribution is

intrinsically large.

Summary: Chapter 2 introduces a coherent study design that allows one to systematically measure

ligand induced effects of agonists, SERMs, and antagonists on the dynamics and structure

of ERα-LBD. This chapter also describes the site directed mutation, production, and

characterization of the proposed mutants using LC-MS and [3H]estradiol binding. The

next Chapter deals with application of the techniques and topics described in both

Chapters 1 and 2. Here we apply SDSL to the mutants produced using the procedure

described in Chapter 2 and analyze the result as part of the goal of integrating existing

information about structure from X-ray data with dynamic information derived from

EPR. Chapter 2 also discusses some important technical concerns regarding plasmid

mutation, protein expression and signal acquisition that have been the focus of

optimization. The next Chapter deals with the application of the techniques so far

described to measure the dynamic response of ER to ligands and coactivators.

55

Chapter 3: Dynamic and structural response of the

estrogen receptor ligand-binding domain to ligand and

coactivator binding

56

3.1 Introduction Estrogen Receptor alpha (ER-α) is a transcription factor belonging to the nuclear

receptor (NR) superfamily that acts to regulate the expression of target genes involved in

development, metabolism and reproduction. ERα transactivation is estradiol dependent

and is directed to genomic estrogen response elements (ERE) by tissue specific co-

activators and co-repressors proteins. Members of the NR family share limited sequence

similarities, however, they are structurally homologous with functionally independent

domains as summarized in Figure 3.1 32,33,43. The full length ER-α is composed of an N-

terminal activation function 1 (AF1) domain, a DNA binding domain, a short hinge

region and the C-terminal ligand binding domain (LBD) where the activation function 2

(AF2) region is located and where selective ligand binding and allosteric protein

interaction occurs. This characteristic architecture makes it possible to investigate the

activity of individual domains separately. Unliganded ER-α is found associated with

chaperone heat shock proteins (HSP90) which stabilize the Apo conformation. Upon

ligand binding, HSP are released and dimers are formed. The general mode of action of

ER-α has been described as a tripartite model where the ligand, dimerized receptor and

specific coregulator complex is necessary to carry out target specific genomic modulation

15.

57

Figure 3.1: Model of ERα-LBD showing ligand induced conformations of helix 12 for agonist (purple, 1ERE) and antagonist (red, 3ERT) ligands (A). Homodimer conformation of ERα-LBD (1ERE) showing helix 12 highlighted in yellow; the labeling positions are marked by spheres, at 543 in blue and at 530 in orange (B). Molecular models were generated with VMD using the Research Collaboratory for Structural Bioinformatics protein data bank (RCSB-PDB) files 1ERE and 3ERT.

In addition to its normal homeostatic functions, ER-α plays a major role in the

progression of estrogen responsive breast cancer and several other disorders such as

osteoporosis, obesity and ovarian cancer 44-46. The importance of ER-α as target of

current breast cancer therapies and as a general model for the action of NRs has attracted

much attention since its initial purification and cloning 13,47.

Structural information available on ER-α up to now has been obtained almost

exclusively from crystallographic studies of ER-LBD bound to ligands with different

A B

58

biological activity 20,48,49. Structures poised in the agonist conformation (e.g. estradiol

bound) differ from antagonist structures mainly in the position of the C-terminal helix 12

(H12). In agonist structures H12 is stabilized in a “closed conformation” which folds over

the binding pocket and forms the AF2 surface where coregulators bind. Ligands with

antagonist character tend to disrupt proper folding of H12 thus inhibiting coregulator

binding which is necessary for ER to carry out its downstream effects. Although crystal

structures have provided valuable insights for elucidating some ligand responses, they

have also been unable to thoroughly explain the action of an important class of ER

binding therapeutic agents known as selective estrogen receptor modulators (SERMs)

50,51. This class of compounds shows promise in clinical applications where SERMs such

as raloxifene and tamoxifen show anti-estrogenic action in estrogen responsive breast

cancer cells while behaving as agonists in other tissues such as the uterus 17. It has been

postulated that SERMs act to partially disrupt the formation of AF2 by affecting the

position of H12, however, the static picture provided by X-ray crystal data has not been

sufficient to explain the complex biological effects of these compounds.

Recently, evidence has emerged pointing to a more active role of allosteric

coregulator binding in defining the structure of ER-α 52,53. Tamrazi et al. used site

specific fluorescent labeling at C530 to probe dynamic changes upon ligand and

coregulator peptide binding providing evidence for a strong stabilizing effect of

coactivator binding on the ER dimer. These finding underline the importance of allosteric

binding as part of the ligand dependent structural remodeling of the receptor. In fact,

59

proper dimer configuration is essential for the downstream transactivation function in the

full length ER-α.

In this chapter we provide direct evidence for dynamic and structural effects of

ligand binding and allosteric coregulator binding on the H12 region of ERα-LBD. Using

site directed spin labeling (SDSL), we introduced an iodomethyl nitroxide spin label

(IMSL) at positions 543 (ERα-LBD C417S, C381S, C530S, M543C) and 530 (ERα-LBD

C417S, C381S) of ERα-LBD 21,34,54. We used electron paramagnetic resonance (EPR) to

monitor local dynamics of the label and double electron-electron resonance (DEER) to

measure structural response of the dimerized receptor to a selected set of ligands and

coregulator peptides. Here, we also report an improved synthetic scheme for production

of IMSL. This label produces a stable thioether linkage and a four bond tether to the α-

carbon. We expect this to make IMSL a more accurate probe of backbone motion

compared with the more widely used methanethiosulfonate spin label (MTSSL) which

produces a five bond tether with an extra degree of rotameric freedom.

The ligand series selected for this study have similar binding modes, but different

biological responses ranging from full agonist to partial antagonist to full antagonist 34-36.

Their common estradiol steroid scaffold, orients the ligand in the binding pocket

conserving the interaction of hydroxyl groups at position 3 with Glu353, Arg394 and at

position 17 with His524 respectively 37. The different activities of the compounds can be

attributed to the presence of side chains at the 11β or 7β positions as in the case of the

antagonist ICI-182-780.

60

We used co-activator derived peptides (D22, SRC-1, TIF-2, RIP-140), containing

the conserved LXXLL motif to investigate the effect of allosteric interaction on the

dynamics of C530 hinge region and on the structure of the ER dimer. These peptides are

known to bind in a ligand dependent fashion to ER-α, and are commonly used as

predictors of a ligands’ biological activity.38,41

3.2 Materials and Methods

(a) Protein Expression, Purification and Spin Labeling The two mutants produced are derived from wild-type ERα ligand-binding

domain (hERα-LBD) (a.a. 302-552) expressed in a pET15b vector (23). The wild type

protein contains four cysteine, three of which (C381, C417, C530) are solvent exposed

and therefore reactive to electrophilic labeling, the fourth (C447) is buried and proven to

be unreactive to labeling 42. The mutant construct C318S/C417S was used to allow the

selective labeling of C530. Production of a singly reactive cysteine at position C543 was

accomplished by mutagenesis of M543C in the cysteine-less construct.

Plasmid mutation was carried out as previously described in Section 2.3.

His tagged ER was purified from the cell lysate with Ni-NTA affinity column

using a flow-through method using standard elution in 300 mM Imidazole. Labeling was

accomplished by incubating the eluate, with typical protein concentration between 10 and

30 mg/ml, with 5 fold mole excess of iodomethyl spin label (IMSL, see below) for a

minimum of 4 hours. Fractions containing the protein were then assayed by SDS-PAGE

electrophoresis, and pooled. A ten fold dilution of the pooled fraction with 50mM Tris,

10% Glycerol at pH 8.1 was used for loading onto a pre-equilibrated cation exchange

61

column. After extensive washing to remove unreacted label, the elution was carried out

with 50mM Tris, 10% Glycerol at pH 8.1 buffer containing 200mM KCl. Samples

prepared by this method were found to be >90% pure when assayed by LC-MS. If

necessary, the sample was further purified with Zeba desalting spin columns (Thermo

Scientific, Rockford, IL) to remove residual spin label.

(b) Ligands The ligand structures used in the study are summarized in Figure 3.2. The ligand

series tested includes agonist, SERM and antagonist ligands: (i) Estradiol (E2) [Estra-

1,3,5(10)-triene-3,17-diol] was purchased from Sigma-Aldrich (St. Luois, MO). (ii) 11β-

2C [Estra-1,3,5(10)-triene-3,17-diol,11-ethenyl-(11β,17β)] 55. (iii) 11β-5C [Estra-

1,3,5(10)-triene-3,17-diol,11-pentyl-,(11β,17β)] 56. (iv) RU 39,411 [Estra-1,3,5(10)-

triene-3,17-diol,11-[4-[2-(dimethylamino)ethoxy]plenyl]-(11β,17β)], 57. (v) RU 58,668

[Estra-1,3,5(10)-triene-3,17-diol,11-[4-[[5-[(4,4,5,5,5-pentafluoropentyl) sulfonyl]pentyl]

oxy]phenyl]-(11β,17β)]. (vi) ICI 182,780 [7β [9]-(4,4,5,5,5-pentafluoropentyl)sulfinyl-

[nonyl]estra-1,3,5(10)triene-3,17β-diol] 58. Compounds ii and iv were prepared by

R.Hanson using the procedures described in the references. Compound iii was prepared

by R. Hochberg using the procedure described in the reference. Compounds v and vi

were provided to R. Hanson by Roussel-Uclaf and Astra-Zeneca respectively.

62

Figure 3.2: Chemical structures of ligands used in the study: (i) Estradiol, (ii) E11β-2, (iii) E11β -5, (iv) RU 39,411, (v) RU 58,668 and (vi) ICI 182,780.

(c) Peptides The coactivator peptides used in this work (Table 3.1) were selected to include the

conserved LXXLL binding motif. All the peptides are known to bind ER-α with ligand

dependent affinity. Steroid Receptor cofactor 1 (SRC-1), receptor interacting protein

(RIP-140), translation initiation factor TIF-2 and random phage display peptide (D22),

were acquired from AnaSpec, Inc (San Jose, CA). The peptide sequences, with the

exception of D22, are derived from naturally occurring coregulator proteins.

O

F F

F

F F

OH

OH

S

(i) (ii) (iii)

(iv) (v) (vi)

OH

OH

OH

OH

O H

O H

N O

O H

O HF

F F F F

O

OS O

OH

OH

63

Table 3.1: Peptide sequences of the peptides used in this study derived from coregulator proteins. The LXXLL motif is shown in boldface. SRC-1 L-T-E-R-H-K-I-L-H-R-L-L-Q-G RIP-140 S-F-S-K-N-G-L-L-S-R-L-L-R-Q-N-Q-D-S-Y hNCoA-2/TIF-2 E-K-H-K-I-L-H-R-L-L-Q-D-S D22 L-P-Y-E-G-S-L-L-L-K-L-L-R-A-P-V-E-E-V

(d) Synthesis of IMSL The iodomethyl spin label employed in this study was synthesized using a new

protocol developed by Adam Hendricks in Prof. Robert Hanson’s laboratory. To 2,2,5,5-

Tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid (389mg, 2.11 mmol) in 7mL of freshly

dried THF was added 0.64 mL of triethylamine (4.55 mmols) and the solution was cooled

to 0oC. Ethyl chloroformate (0.30 mL, 3.14 mmols) in 3mL of THF was added dropwise

over 30 minutes and stirring continued at 0oC for another 45 minutes. The reaction

mixture was filtered to remove triethylamine-hydrochloride, and sodium borohydride

(113 mg, 2.99 mmols) in 5mL of water was added over 20 minutes, while maintaining

0oC. After one hour the reaction was stopped and 1N HCl was added slowly to quench

excess sodium borohydride. The solution was extracted with dicloromethane and washed

with brine (3x 30mL). The organic layer was dried over magnesium sulfate. Upon

removal of the magnesium sulfate, florisil was added and the mixture was evaporated.

Purification by flash chromatography (70/30 hexane/ethyl acetate) yielded 220 mg of

yellow crystals, 61.2% yield. IR; 3400cm-1 O-H, 2950 cm-1 alkyl C-H, no carbonyl peaks

present. MP: 74-76oC,

64

To 2,2,5,5-Tetramethyl-3-pyrrolin-1-oxyl-3-ol (48 mg, 0.282 mmol) in

dichloromethane (10 ml) was added triethylamine (0.1 mL, 0.712 mmol), and the solution

was cooled to -10ºC. To this solution methanesulfonyl chloride (0.1mL, 1.283 mmol) was

added and the reaction was stirred for 10 minutes at -10ºC and then overnight at room

temperature. The reaction solution was partitioned between saturated sodium chloride

and dichloromethane. The organic layer was separated and dried over magnesium sulfate,

filtered and evaporated to dryness. The residue was purified via flash chromatography,

(70/30 hexane/ethyl acetate), yielding 49 mg of yellow/green crystals (70% yield). IR:

2989 cm-1 alkyl C-H, 900cm-1 alkene =C-H, 1380 cm-1 and 1135 cm-1 sulfone SO2. MS:

M+2 250.04 100%. MP: 83-84oC.

(e) Mutant characterization Determination of binding affinities (Kd) of spin labeled mutants was carried out by

saturation binding curves using [3]-estradiol in Prof. Katzenellenbogen lab at the

University of Illinois, Urbana-Champaign using previously described methods.59 Protein

activity for spin labeled mutants was determined from [3H]estradiol binding and protein

concentration was estimated using the Bradford assay.60 Purity and molecular weight

were determined from SDS-PAGE and LC-TOF-MS. The results of these analyses are

summarized in Table 3.2. The percent activity reported in Table 3.2 was calculated based

on the molar ratio of 3H-estradiol to receptor in the fully saturated case; in this respect

100% activity represents the case where every receptor present is occupied by a ligand

molecule. It should be noted that even under optimal conditions only about 80% of the

receptor is available for binding. Our results show slightly lower activity ranging from 68

65

to 62% which might be a result of exposure to temperature variations during transport as

well as freeze-quench cycles.

Table 3.2: Summary of the ERα-LBD mutants produced and characterization by TOF-MS and [3H]estradiol binding. Mutant name

Construct Label position Observed mass (Target mass)

Binding affinity (% activity)

ER-543 C381S,C417S, C530S,M543C

543 30748.9 Da (30748 Da)

1.2 nM (68 %)

ER-530 C381S,C417S 530 30792.2 Da (30792 Da)

0.85 nM (62%)

(f) Incubation of labeled receptor with ligand/coactivators: The labeled receptor was incubated under saturating condition for both the ligand and the

coregulator peptide studies. A 2 mM stock solution of the steroid ligands in 1:1

water:DMF solvent was prepared by dissolving the appropriate amount of ligand in 100%

DMF to which water was added. Coregulator stock solutions were prepared by

reconstituting the lyophilized peptides to a final concentration of 1 mM in 20 mM Tris,

pH 8.1. For each mutant, all the measurements reported here are derived from the same

stock of protein. This ensures that the amount of inactive or unlabeled protein is the same

in each experiment, making the changes observed dependent to ligand-receptor

interactions only.

(g) CW-EPR spectroscopy CW-EPR measurements were conducted on a Bruker EMX instrument equipped with

high-sensitivity cylindrical cavity. All spectra were acquired at room temperature at a

66

microwave frequency of 9.37 GHz, 0.2 mW microwave power, with 0.8 G 100 kHz field

modulation amplitude. Each spectrum reported here results from a 30 scan average.

Ligands and coregulators were allowed to bind under saturating condition (3 to 5 fold

molar excess of ligand to protein) for a minimum of 30 min at 4 oC before EPR

measurement.

(h) DEER Spectroscopy For DEER experiments, a 30% glycerol buffer was used for both spin-labeled ER-543

and ER-530 mutants. This buffer ensures glass formation, which provides homogeneous

protein distribution and prevents phase separation induced by micro-crystallization of

water during freezing. Samples (100 μL) in 4 mm O.D. 3.2 mm I.D. quartz capillaries

were fast frozen by dipping into liquid nitrogen immediately before inserting into the

precooled resonator. Pulsed EPR spectra were recorded on a Bruker Elexys 680

spectrometer equipped with a Bruker dielectric ring resonator (model MD-5). In the 4-

pulse DEER experiment 61,62, the frequency of the pump pulse was adjusted to the

resonator dip and the static field was set to the low-field resonance of the nitroxide signal.

To minimize the orientation selection and maximize the fraction of coupled spins (and

thus signal-to-noise), the observed pulse was applied at 65 MHz upfield from the pump

frequency. This arrangement observes the spins at the low field resonance while pumping

the spins at the center of the spectrum. Typical π/2 pulse lengths were 16 ns, and the echo

was integrated using an integration window equal to the echo width. Data were analyzed

using home-written MATLAB software, which assumes a sum of Gaussian-distributed

conformers contributing to the observed echo modulation.

67

The program employs the Monte Carlo/SIMPLEX algorithm to find the distance

distribution resulting in the dipolar evolution function that best describes the

experimental data 63. Selection of the number of Gaussian populations used to describe

the experimental data is based on a statistical F test. The software is available at

http://fajerpc.magnet.fsu.edu.

3.3 Results

(a) Selection of labeling sites The two ERα-LBD mutants constructed have been designed to allow site directed

spin labeling at positions expected to undergo ligand induced changes as predicted by X-

ray structures. Because dynamics of H12 are particularly important to explain

ligand/receptor/coregulator interaction, this domain was labeled directly by placing the

IMSL label at position 543. From the crystal structures available, this position may be

expected to undergo substantial dynamic changes that reflect a transition from the

motionally constrained local environment of the agonist conformation to less constrained

environment in the antagonist state. A [3H]estradiol binding assay shows retention of

high binding affinity for spin labeled ER-543 (1.2 nM compared to 0.11 nM for wild

type) indicating that labeling of position 543 does not severely affect receptor-ligand

interaction.

The hinge region between H11 and H12 at position 530 has been previously

studied using fluorescence labeling and, in one case, spin labeling.64 These studies

demonstrate that position 530 is a good reporter of both ligand binding and dimer

stability. ERα-LBD was therefore spin-labeled at position 530. The label at this position

68

was studied using CW-EPR to investigate local dynamic changes in response to ligand

binding and allosteric coregulator interaction, and DEER spectroscopy to measure the

structural response of the dimer conformation to ligand and coregulator interaction. A

[3H]estradiol binding assay for spin labeled ER-530 confirms retention of high binding

affinity to estradiol with a Kd of 0.9 nM.

(b) Ligand induced dynamic changes of H12 Site directed spin labeling of H12 was carried out on the ERα-LBD mutant ER

543 (C381S/C417S/C530S/M543C) to measure the dynamic effects of ligand binding on

this region by CW-EPR. Mutations C381S, C417S and C530S were necessary to remove

the three solvent exposed cysteines naturally present in the ER-LBD sequence, in

addition we introduced mutation M543C to provide a uniquely reactive cysteine on H12.

Based on available mutational and crystal data, position 543 is expected to tolerate

labeling with IMSL while retaining all the necessary interactions with the ligand; on the

other hand, the IMSL sidechain at this position may interfere substantially with

coregulator recruitment on the AF2 surface. For this reason, the ER-543 mutant was used

only to study ligand binding interactions.

ER-543 proved to be a sensitive reporter of H12 dynamics in response to ligand

activity. The EPR spectra obtained from this mutant, summarized in Figure 3.3, show a

trend of higher mobility with increasing antiestrogen character of the ligand. The agonist

ligands Estradiol and E11β-2 produce very similar spectra that show lineshape features

characteristic of strong immobilization. This is consistent with crystal structures of ER in

the agonist state, in which the label experiences substantial interaction with nearby

69

sidechains. Strikingly, the Apo-ER exhibits similar immobilization, suggesting that the

unliganded receptor exists in a rather tightly folded state.

Figure 3.3: EPR spectra summarizing the effect of ligands on dynamics of ER-543. From the top: Apo, Estradiol, E11β -2, E11β -5, RU 39,411, RU 58,668, ICI 182,780.

Ligands with progressive substitutions at the 11β position of estradiol show direct

effects on the mobility of H12. The five carbon alkyl chain in E11β-5 produces lineshape

changes that suggest an increase in mobility when compared to agonist ligands. This

trend continues with the antagonist ligands RU 39,411 and RU 58,668, which produce

spectra consistent with less rigid motion. Finally, the classic antagonist ICI 182,780

3470 3480 3490 3500 3510 3520 3530 3540 3550 3560

ICI 182,780

RU 58,668

RU 39,441

E11-5

E11-2

Estradiol

Apo

Magnetic Field (Gauss)

Liga

nd

70

yields similar dynamic effects to the antagonist RU 58,668 confirming that mobility of

H12 is correlated with ligand activity.

Changes in probe mobility were evaluated from characteristic lineshape features

of the spectrum. Specifically, the mobility parameter ΔH0−1 was calculated from the

inverse of the width of the central resonance line. The results from this simplified

analysis of the label dynamics are plotted versus ligand in Figure 3.4. A more

comprehensive analysis of the diffusion and order parameters using NLS lineshape fitting

program was inconclusive in producing satisfactory fits to this data. This is not surprising

when we consider that the spectra are composed of multiple components and that up to

40% of the labeled receptor might be inactive and therefore not affected by ligand

binding. Nonetheless, the changes in spectral lineshape are supported by the semi

quantitative ΔH0−1 parameters.

Figure 3.4 shows that agonist ligands such as Estradiol and E11β-2 result in a

high degree of immobilization that surprisingly is reflected in the Apo state as well. In the

case of E11β-5 and RU 39,411 the spectra show gradual changes that are consistent with

an increase in the local dynamics as reflected by ΔH0−1. Finally, the full antiestrogens RU

58,668 and ICI 182,780 provide the highest degree of ligand induced mobility.

71

Figure 3.4: Mobility parameter ΔH0

−1 derived from EPR spectra of ER-543 in the presence of the selected ligands.

(c) Dynamic and structural changes of H12 hinge region in response

to ligand-coregulator interaction

Ligand/coregulator induced dynamic changes of the hinge region of H12 provide

important insights into the structural basis of ER action. Site directed labeling at the C530

position of the hinge region can be conveniently achieved by mutating the solvent

exposed C417 and C381 residues to serine, leaving a single labeling site on the native

cysteine at position 530. Crystallographic data of agonist and antagonist conformations

shows that this hinge region is relatively unstructured but it is expected to undergo

Apo Estradiol E11-2 E11-5 RU 39,411 RU 58,668 ICI 182,7800.2

0.25

0.3

0.35

0.4

0.45

Ligand

ΔH

0-1

72

conformational and dynamic changes that reflect the action of ligand on the relative

position of H12. Previous studies report with retention of wild type like binding

characteristics when either fluorescence or spin labels are attached at position C530 of

ER-LBD. The activity of ERα labeled at position 530 by IMSL was confirmed by

measuring its binding affinity to estradiol.

Spin labeled ER-530 was allowed to bind to each ligand under saturating

conditions. Figure 3.5 summarizes the EPR spectra for the ER-530 bound to each ligand

in the series tested. The unliganded ER-530 Apo shows a higher level of local mobility

compared to ER-543 Apo. Ligand binding causes subtle changes in the EPR lineshape of

ER-530. As one follows the series of ligands from estradiol to E11β-2 and E11β-5 one

first notices lineshape changes consistent with the appearance of a less mobile

component. This effect is observable in the spectral region between 3480 and 3500 Gauss

from the presence of a broad shoulder feature. The antiestrogen character of RU 39,411

produces an increase in local mobility, while both RU 58,688 and ICI 182,780 yield

complex spectra with both slow motional and fast motional components.

73

Figure 3.5: EPR spectra summarizing the effect of ligands on dynamics of ER-530. From the top: Apo, Estradiol, E11β -2, E11β -5, RU 39,411, RU 58,668, ICI 182,780.

Coregulator/ligand binding effects on dynamics of the H12 hinge region was

investigated for four coregulator peptides in the presence of the ligands. EPR spectra

obtained from ER-530/ligand/SRC1 are summarized in Figure 3.6 and show

representative changes in the dynamic modulation of the hinge region in the presence of

ligand and peptide. Changes in the observed dynamics of ER-530 in the presence of

coregulator peptides are evidence of allosteric interaction of the ER/ligand complex with

coregulator peptides. The Apo, estradiol and E11β-2 cases show broader lineshape

features consistent with decreased mobility in the presence of coregulator peptides. As

3460 3480 3500 3520 3540 3560 3580

ICI 182,780

RU 58,668

RU 39,441

E11-5

E11-2

Estradiol

Apo

Magnetic Field (Gauss)

Liga

nd

74

one progresses though the series towards ligands with a more antiestrogen character, the

degree to which the peptide induces dynamics changes of the hinge region decreases

markedly especially when comparing spectra of ER/ligand and ER/ligand/peptide. The

spectra obtained with the antiestrogens RU 39,411, R58,668 and ICI 182,780 show that

peptide has a much smaller effect on lineshape when these antagonist ligands are present,

consistent with the notion that antagonist ligands prevent coregulator peptide recruitment.

Interestingly the SERM E11β-5 produces intermediate changes to those of estradiol and

the antiestrogens. This validates the model of partial agonist for this class of compounds

and provides evidence for an allosteric effect on the receptor’s dynamics. In fact, current

evidence suggests that SERM ligands induce selective binding of coregulators, thus

causing different biological effects in tissues with different coregulator profiles.

Figure 3.7 shows ΔH0−1 mobility parameter computed for both ER/ligand and

ER/ligand/SRC1. The results suggest a decrease in mobility upon addition of peptides

that correlates with the already mentioned lineshape changes. In the presence of the

antiestrogenic ligand RU 39,411, coregulator peptides produce no change in ΔH0−1 and

no difference in lineshape suggesting that peptide binding is inhibited.

75

Figure 3.6: EPR spectra summarizing the effect of ligands + peptide (SRC1) on dynamics of ER-530. From the top: Apo, Estradiol, E11β -2, E11β -5, RU 39,411, RU 58,668, ICI 182,780.

3440 3460 3480 3500 3520 3540 3560 3580 3600

ICI 182,780 + SRC1

RU 58,668 + SRC1

RU 39,411 + SRC1

E11-5 + SRC1

E11-2 + SRC1

Estradiol + SRC1

Apo + SRC1

Magnetic Field (Gauss)

Liga

nd

76

Figure 3.7: Mobility parameter ΔH0−1 derived from EPR spectra of ER-543 in the

presence of the selected ligands (○) and ligands plus SCR-1 peptide (▼).

EPR spectra can be directly correlated to ligand activity by comparing the

cumulative relative squared difference (RSD) between the normalized spectra of

ER/ligand and ER/ ligand/peptide for each of the four coactivator peptides studied. This

approach is justified by the fact that the spectral changes observed in the presence of

peptide give rise to similar lineshape effects that can be compared relative to each other

65. We interpret RSD values as a measure of peptide induced change of dynamics where

Apo Estradiol E11-2 E11-5 RU 39,441 RU 58,668 ICI 182,7800.3

0.35

0.4

ΔH

0-1

Ligand

77

higher RSD values correspond to larger changes and low RSD values correspond to small

changes.

Figure 3.8 shows the RSD values computed for the four peptides studied as a

function of the ligand present. The agonist ligands estradiol and E11β -2 consistently

show higher RSD values compared to the antagonist ligands RU 39,411, RU 58,688 and

ICI 182,780 or the unliganded Apo state. In the case of the SERM E11β-5 the RSD

values are intermediate to those from estrogenic and antiestrogenic compounds and

appear to be quite sensitive to the identity of the coregulator peptide. This observation

supports a model in which SERMs act to differentially disrupt coregulator binding.

Figure 3.8: RSD values computed between spectra of ER-530/ligand and ER-530/ligand/peptide. RSD values are calculated from Σ√(Ia-Ib)2, where Ia and Ib are the spectral intensities of the normalized and aligned spectra at every point.

0

0.1

0.2

0.3

0.4

0.5

0.6

Apo Estradiol E11-2 E11-5 RU 39,411 RU 58,668 ICI 182,780

RIP-140 TIF-2SRC-1 D22

Ligand

RS

D

78

DEER spectroscopy was performed to investigate whether the dynamic changes

noted above arise from structural differences brought about by ligand/coregulator

interaction. ER-530 was examined in the Apo state, bound to Estradiol, and in the

presence of both Estradiol and D22 peptide. In the case of singly labeled ER, DEER

spectroscopy will reflect interspin distances between position 530 on each of the two

halves of the ER dimer. Figure 3.9 summarizes the DEER spectra and the analysis 62. We

find that the combination of Estradiol and coregulator binding has a marked effect on the

structure of the dimer by producing changes in both the distance and the distance

distribution between the halves of the dimer in comparison with the Apo state. More

precisely, the mean distance changes from 30 Å in the Apo and 27 Å in the Estradiol

bound state to 22 Å in the presence of estradiol/D22, while the distance distribution

measured as the half height width of the Gaussian distribution goes from 15 Å to 4.5 Å.

The distance measurements are consistent with both the crystal structures as well as

FRET measurements that show a pronounced stabilization of the dimer structure upon

ligand and peptide binding. These results provide, for the first time, quantitative

measurements of the solution structure of ER dimers and suggest that dynamic changes

described arise from conformational re-modeling of the receptor brought about by ligand

and coregulator interaction.

79

DEER Spectrum Distance Distribution Error Analysis

0.5 1 1.50

0.2

0.4

0.6

0.8

1

t (µs)

Nor

m. I

nten

sity

0 2 4 6 8

r (nm)2 4 6 8

0

1

2

3

4

5

r (nm)

wid

th (n

m)

0.5 1 1.5

0

0.2

0.4

0.6

0.8

1

t (µs)

Nor

m. I

nten

sity

0 2 4 6 8r (nm)

2 4 6 80

1

2

3

4

5

r (nm)

wid

th (n

m)

0.5 1 1.5

0

0.2

0.4

0.6

0.8

1

t (µs)

Nor

m. I

nten

sity

0 2 4 6 8r (nm)

2 4 6 80

1

2

3

4

5

r (nm)

wid

th (n

m)

Figure 3.9: DEER spectra and analysis of ER-530 Apo (top), ER-530/estradiol (middle) and in the ER-530/estradiol/D22 (bottom).

3.4 Discussion Site-directed spin labeling has been applied in this study to measure local

dynamic and structural changes that accompany ligand binding and coregulator peptide

interaction with ER-α LBD. Additionally, we describe an EPR based method to measure

changes in the local dynamic of H12 hinge region that are specific to ligand/coregulator

80

binding. The ligands series selected for this work is designed to systematically test the

effect of substitutions on the estradiol scaffold on H12 dynamics.

Recent work by Dai et al. using Hydrogen Deuterium exchange Mass

Spectrometry (HDX-MS) to measure ligand induced dynamic changes in ER-LBD has

shown that receptor dynamics is an accurate indicator of ligand character 66. Interestingly,

the study reported no change in the dynamics of H12 as a function of binding of ligands

with different biological activities, in contrast to the results reported here, where dynamic

effects of ligand binding on H12 are clearly evident. A consideration of how dynamics is

measured in HDX versus EPR can reconcile these two observations. HDX relies on

differences in the rate of Hydrogen-Deuterium (H-D) exchange in different

conformations of the receptor, which is mainly affected by solvent exposure. Increased

dynamics will likely lead to increased solvent exposure; however, a surface helix such as

H12 is already substantially exposed to the solvent, and it is unclear whether changes in

the backbone flexibility at such a location are observable by this technique. In contrast,

EPR spectra are sensitive to both local environment and backbone flexibility on the ns

timescale. Combining these two observations, the picture that emerges of H12 is that of a

domain with high level of solvent exposure, demonstrated by the fast rate of H-D

exchange, and with a ligand dependent behavior consistent with progressive

destabilization of the backbone structure upon binding of agonist, SERM or antagonist

ligands.

EPR spectra of ER-543 proved to be very sensitive reporters of H12 dynamics,

and point to a significant destabilization of the helix structure in the antagonist

81

conformation. Since spin probe dynamics is affected to a great extent by the local

secondary structure, the dynamic changes observed may be interpreted as reflecting the

combined effects of backbone fluctuation as well as domain motions. IMSL is a

particularly sensitive probe of backbone dynamics due to its shortened tether to the alpha

carbon. The changes observed in ER-543 suggest that the secondary structure of H12 is

directly affected by ligand interaction, so that binding of antiestrogen ligands with long

hydrocarbon chains have a disrupting effect on the helical conformation of H12. The EPR

lineshape is reflective of both local environment and internal rotameric state of the

nitroxide probe. The changes we observe in ER-543, going from an immobilized state to

a more dynamics state, may arise from a change in the local environment where the label

experiences a less stericly hindered motion with antagonist ligands. Alternatively, the

change may be explained by disruption of the secondary helical structure that affects the

interact dynamics of the label through increasing flexibility of the alpha-carbon. The

results further demonstrate that this effect can be controlled by incrementing the length of

hydrocarbon substitutions at the 11β position of estradiol. In essence, the ERα-LBD spin

labeled at position 543 acts as a molecular probe of ligand-receptor interactions that

differentiates agonist, antagonist and SERM ligands based on their effect on H12.

Ligand binding is not the only determinant of ER action, binding of tissue specific

coregulator proteins to AF2 is just as essential to the downstream biological effects of ER

is. Recently, it has become apparent that ligand binding alone is not sufficient to

“commit” H12 to an agonist conformation but allosteric coregulator interaction plays an

important role. We used ER-530 to probe the effects of ligand and coregulator binding on

82

dynamics of the hinge region and on the structure of the ER homodimer. Position 530 has

been extensively targeted for site directed labeling using mainly fluorescent probes but

also spin probes 53,64,67. These studies have been among the first to demonstrate ligand

dependent effects of ligands on ER dynamics. In the context of labeling of position 530,

our investigation is designed to extend the current model by providing measurements of

both ligand and coregulator induced dynamics as well as distance measurements on the

ER dimer.

The hinge region is indirectly affected by ligand binding, but still produced

characteristic changes observable by EPR. A clear stabilizing effect is observed when

both agonist ligands and coregulator peptides are present. We correlated peptide induced

changes with ligand activity and found a trend of motional stabilization for agonist

ligands and lower stabilization of antagonist ligands. Interestingly, the SERM E11β-5

falls between agonist and antagonist and shows peptide specific effects. This observation

allows us to formulate a definition for SERMs that is based on precise effects on the

structure of ER-LBD that results in discrimination in binding among different coactivator

peptides.

The results establish that the stability of the ER-LBD dimer is affected by ligand

binding. DEER measurements of ER530 in the presence of estradiol and coregulator

peptide provide structural evidence to support the dynamic changes observed. Although

we observe a slight enhancement in the stabilization of ER-LBD dimer by agonist

ligands, the most dramatic effect occurs upon allosteric binding of coactivator peptides,

which produces a more compact and ordered dimer structure. This is the first application

83

of DEER spectroscopy to ER-α and as such it may provide insight into the dynamics of

other structurally analogous NRs. In light of these results, ER-α response can be

considered in terms of an order-disorder transition, in which successive ordering

interactions define the biological effect of the receptor.

Although little structural information is available about the unliganded state of

ER-α, the spin-labeling results for Apo-ER contrast with conclusions from structural

studies of other unliganded NRs. The crystal structure of the unliganded nuclear receptor

peroxisome proliferator-activated receptor γ (PPAR-γ) and NMR data on PPARα in both

the Apo form and in complex with ligand shows an overall stabilization of the LBD upon

agonist binding, 68,69. Other studies on similar nuclear receptors, such as the Retinoid X

Receptor (RXR), generally point to an Apo conformation with a substantial degree of

flexibility that undergoes a transition to a more compact structure following ligand

binding 70 . In this study, we observed only minor changes in local dynamics due to

ligand binding at positions 543 and 530. The Apo spectra on both cases closely resemble

the spectra obtained when agonist ligands are present, displaying a constrained

environment at position 543 and more flexible dynamics at position 530. This

observation is also confirmed by the DEER results, where only small differences appear

between Apo and estradiol bound receptor dimer. Despite the similar dynamics, the

agonist bound state yields a more pronounced coregulator induced effect (Figure 3.8)

compared with the Apo state. When these observations are taken together they suggest a

system where the agonist ligand acts to narrow the conformational space of the Apo state

to optimize coregulator binding. As such, the action of the ligand is not only to stabilize

84

the local structure, but more importantly to “prepare” the receptor for coregulator

interaction by favoring the most effective conformation.

3.5 Conclusions The results presented here demonstrate that (i) binding of ligands to ERα-LBD

induce dynamic changes in H12 and in H12 hinge region that directly correlate with the

ligand’s biological activity and (ii) ligand dependent allosteric binding of coregulator

peptides induces dynamic and structural remodeling effects on ERα-LBD. We have used

SDSL and EPR to extend the current model of ERα-LBD action providing dynamic

information on H12 region and quantitative structural information on the dimer

conformation.

In addition to providing new dynamic and structural information about ligand-

receptor interaction, we believe that SDSL applied to ER-LBD can be used as a predictor

of ligand activity and coregulator interaction. Our results show that it is possible to

discriminate agonist, antagonist and SERM ligands with a simple comparative analysis

which are based on the physical response of the receptor to ligand binding. Indeed, a

major finding of this study is that SERMs differentially affect coregulator binding, in a

manner that apparently depends upon the specific recognition sequence of the

coregulator.

Other biologically important members of the NR superfamily such as ERβ, the

Thyroid Receptor and the Androgen Receptor share similar structure and mode of action

with ERα suggesting that the mechanisms here described are likely to represent a

significantly wider group of NRs. Although we have measured ligand induced dynamics

85

of H12, questions still remain about the nature of the structural changes taking place. To

what extent increase in dynamics reflects movement of H12 as a “rigid body” versus

unraveling of the helical domain? What is the effect of non-steroidal ligands? These

questions are relevant to a basic understanding of ligands-receptor interaction and as such

can have importance for the design of therapeutic ligands.

In the final Chapter of this thesis we will explore some ongoing work aimed at

addressing these important questions. In particular we present a new model of

ligand/coregulator induced NR remodeling and discuss the use of double labeled ER to

map more accurately the position and dynamics of H12 with respect to the receptor’s

body.

86

Chapter 4: Characterization of Spin labeled Estradiol

87

4.1 Introduction The steroid estradiol (E2) is responsible for critical physiological functions such as

growth, development and maintenance of many different tissue types (1,2). These effects

are carried out via ligand-activated nuclear receptors known as estrogen receptors (ER),

which function as targeted regulators of gene expression (3).

In addition to carrying out vital homeostasis functions, ER-α is known to

participate in the development and proliferation of estrogen responsive breast cancer as

well as other disease states such as osteoporosis, obesity, and infertility 33,71-74. The

clinical relevance of ER has caused intense interest in the synthesis of small molecules

that can antagonize E2 binding. Many high affinity estrogen derivatives have been

synthesized that can induce agonist, antagonist and partial-antagonist biological

responses. Steroidal derivatives have also been synthesized to provide ER selective

imaging agents for diagnostic purposes. Some of the modifications to the basic estradiol

scaffold include addition of radioactive labels,75,76 IR-active groups,75 organometallic

complexes77 and various fluorophores78,79. Recently the synthesis of a new class of spin

labeled estradiol derivatives was reported where stable radicals in the form of nitroxide

moieties were introduced at the 17β and 16 position on the estradiol scaffold.80 No ER

binding data has been published on these compounds so far, however they are expected to

produce ER selective probes with unique properties such as EPR activity, fluorescence

quenching, sensitivity to local oxidative stress, and enhanced magnetic relaxation of local

environments.

In this chapter we report the EPR, photophysical, and receptor binding

characterization of two previously described nitroxide functionalized estradiol

88

derivatives: HO-2105 and HO-2447 [1-Pyrrolidinyloxy, 2-[(17α)-3,17-dihydroxy-19-

norpregna-1,3,5(10)-trien-20-yn-21-yl]-2,5,5-trimethyl- (9CI)] (Figure 4.1) 80. Binding of

the spin labeled estradiols (SLE) to ERα-LBD was characterized by EPR and intrinsic

tryptophan fluorescence quenching (ITQ). We also investigated photophysical effects of

the nitroxide on E2’s intrinsic fluorescence. DEER spectroscopy was used to measure

interspin distances of bound probes in the ERα-LBD dimer complex providing solution

structure measurements of the homodimer complex. The results presented here offer a

first look at biophysical applications of this unique class of functionalized estradiol.

Figure 4.1: Structures of spin labeled estradiols

4.2 Methods

(a) Protein preparation: ERα ligand-binding domain (hERα-LBD, a.a. 302-552) was expressed in a pET15b

vector (23). Expression and purification procedures are described in Section 2.3.

Briefly, His tagged ER was purified from the cell lysate with Ni-NTA affinity

column by a flow through method using standard elution in 300 mM imidazole. Fractions

containing the protein were then assayed by SDS-PAGE electrophoresis, and pooled. A

ten fold dilution of the pooled fraction with 50mM Tris, 10% glycerol at pH 8.1 was

OO H

OH

N

O

OH

OHN HO-2105 HO-2447

89

necessary for loading onto a pre-equilibrated cation exchange column. After extensive

washing with equilibrate buffer to remove excess imidazole, the elution was carried out

with 50mM Tris, 10% glycerol at pH 8.1 buffer containing 200 mM KCl. Samples

prepared by this method were found to be >90% pure when assayed by LC-MS.

(b) CW-EPR measurements: Continuous wave EPR spectra were obtained using a Bruker EMX spectrometer equipped

with a high sensitivity cylindrical cavity with the following spectral acquisition

parameters: 9.8 GHz frequency, 0.5 Gauss modulation field, 100 kHz modulation

frequency and 20dB attenuation (0.2 mW power). The spectra shown here are averages of

no less than 10 scans.

(c) Fluorescence quenching assay: The assay was carried out in a 50 nM solution of the purified ERα-LBD stock.

Tryptophan fluorescence emission was monitored between 300 nm and 500 nm, while the

excitation wavelength was held at 280 nm. Aliquots of the ligand stock were added to the

protein solution to produce a 1 nM increase in concentration of ligand with every

addition. The sample was mixed by pipetting and allowed to incubate at room

temperature for at least 5 minutes before measuring the fluorescence.

(d) EPR assay Stock solutions of 5 mM HO-2105 and HO-2447 were prepared by dissolving appropriate

amounts of the purified powder in dimethylformamide. Stock solutions of 300 µM ERα-

LBD were purified as described and the concentration was estimated by Bradford assay.

If necessary, protein concentration was performed with Amicon diafiltration cells using a

90

10 kDa molecular weight cut-off membrane to obtain concentrations of about 300 µM

ERα-LBD as estimated by Bradford assay. 1:2 serial dilutions of ERα-LBD stock to a

final volume of 50 µl were performed in 50mM Tris, 10% Glycerol, 150 mM KCl at pH

8.1. Reaction mixtures were spiked with 1 µl of ligand stock to give a final concentration

of 100 µM ligand. Ligand concentration in the reaction mixture was confirmed by spin

counting against a TEMPO standard curve. Reaction mixtures were incubated for a

minimum of 30 min at 4o C and allowed to equilibrate to room temperature prior to EPR

measurements. All measurements were carried on the same protein stocks in order to

account for presence of inactive protein equally.

(e) DEER measurements: Low temperature (65K) DEER spectra were recorded on a Bruker Elexsys 680 with a

Bruker dielectric ring resonator (Bruker, ER 4118X-MD5). 30 wt% of sucrose (final

concentration) was added to the sample to avoid water crystallization and ensure a

homogenous protein distribution. Samples were then transferred into a 4 mm OD/3.2 ID

quartz capillary and snap frozen in liquid nitrogen prior to insertion into the precooled

resonator. The dielectric resonator was overcoupled (low Q factor) to obtain a broad

resonator resonance line and to minimize the interference between the ringing from the

pulses and the signal. DEER was measured using a 4-pulse sequence to provide for the

accurate determination of distance distribution in the range of 2.0-7.0 nm, as described

previously62. The frequency of the pump pulse was adjusted to the resonator dip, and the

observer pulses were applied at 73 MHz lower than the pump frequency. Observing the

spins at the low field resonance while pumping the spins at the center of the spectrum

91

minimizes the orientation selection and maximizes the fraction of coupled spins. Typical

π/2 pulse lengths were 16 ns and π ELDOR pulse length was 32 ns. The echo was

integrated using a window equal to the echo width. Data were analyzed using home-

written MATLAB software, which assumes a sum of 1-4 gaussian-distributed distance

populations contributing to the observed echo modulation. The program employs the

Monte Carlo/SIMPLEX algorithm to find the distance distribution resulting in the dipolar

evolution function that best describes the experimental data. Selection of the number of

Gaussian populations used to describe the experimental data is based on a statistical F

test. The software is available at http://fajerpc.magnet.fsu.edu.

4.3 Results

(a) EPR characterization EPR spectra of nitroxides are sensitive to rotational diffusion rates of the order of

ns. Aqueous solutions of the SLE exhibit a three line spectrum characteristic of fast

tumbling rotation on the ps timescale that averages the anisotropic contributions of the

magnetic g value and hyperfine coupling. Figure 4.2 summarizes the spectra of HO-2105

and HO-2447 in the absence and in the presence of purified ERα-LBD. The lineshape

changes are consistent with going from a freely tumbling state to an immobilized state in

the presence of receptor, demonstrating that both ligands bind to ERα-LBD.

92

Figure 4.2: EPR spectra of HO-2105 (top), HO-2105 in the presence of molar excess of ERα-LBD (bottom), (i). HO-2447 (top) and HO-2447 in the presence of molar excess of ERα-LBD (bottom), (ii). Spectra are an average of 10 scans in 50 mM Tris, 150 mM KCl, 10% glycerol, pH 8.1 at 298K.

We used non-linear least squares lineshape analysis to fit motional parameters to

spectra of spin labeled estradiol bound to ERα-LBD. The resulting fits are shown in

Figure 4.3 and the motional parameters are summarized in Table 4.1. The microscopic

order macroscopic disorder model (MOMD) was used to correlate local dynamics

experienced by the probes with overall tumbling of the receptor ligand complex 22,23.

Although the two ligands are similar, lineshape features suggest differences in their local

motions. HO-2447 in the bound state displays a faster rotational diffusion rate as well as

more isotropic motion (N = 0.98) compared to HO-2105. These differences reflect details

about the local environment of the nitroxide. In HO-2447 the nitroxide is tethered to the

17α position by a propargyl tether while in HO-2105 a more rigid double bond forms a

direct connection between the five-membered nitroxide ring and the estradiol scaffold.

The differences in local mobility can be explained in terms of both local environment and

intrinsic mobility.

3420 3440 3460 3480 3500 3520 3540 3560 3580 3440 3460 3480 3500 3520 3540 3560 3580 3600

(i) (ii)

93

Figure 4.3: EPR spectrum of ERα-LBD bound HO-2105 (gray) and calculated lineshape fit (dashed line), (i). EPR spectrum of ERα-LBD bound HO-2447(gray) and calculated lineshape fit (dashed line), (ii).

Table 4.1. Magnetic and motional Parameter derived from the EPR lineshape fits. Ligand gx gy gz Ax Ay Az R║ N c20 c22 Dα Dβ HO-2105 2.010 2.004 2.002 4.3 5.0 35.8 7.67 0.42 0.82 1.53 22.0 117.9HO-2447 2.008 2.006 2.002 5.0 5.5 35.7 7.85 0.98 1.57 1.06 8.9 142.9

(b) EPR based binding assay In the previous section we have demonstrated that EPR spectroscopy can easily

discriminate between bound and unbound states of the estradiol derivatives HO-2105 and

HO-2447. Here we describe a simple method to evaluate binding affinity

spectroscopically. Figure 4.4 shows EPR spectra resulting from titration of ERα-LBD in

individual solutions containing HO-2105 and HO-2447. By keeping the amount of spin

probe constant and varying the concentration of receptor, we account for exchange

effects that arise at high spin concentrations and alter the lineshape features. The bound

and unbound components can be estimated by two sites NLS fitting 22,23 or, more directly

by measuring the peak to peak intensity (I+1) of the sharp component of the spectrum as a

3260 3280 3300 3320 3340 3360 3380 3400 3420 3440-6 -4 -2 0 2 4 6 x 10 4

3420

3440 3460 3480 3500 3520 3540 3560 3580-3

-2

-1

0

1

2

3x 10 5

(i) (ii)

94

function of receptor concentration. Figure 4.5 was constructed by plotting the difference

in intensity between I+1 in the absence and presence of receptor (I+1,0− I+1,[ER] ) versus

receptor concentration ([ER]). Binding affinities (Kd) for the two compounds were

obtained by fitting the following two state saturation binding curve to the data:

][][max,1

],[1 ERKERI

Id

ER +×

= ++ Eq. 4.1

Using this method we measured binding affinities of Kd = 5.9 μM for HO-2105, and 16.7

μM for HO-2447.

Figure 4.4: (i) EPR spectra of HO-2105 titrated with ERα-LBD. (ii) EPR spectra of HO-2447 titrated with ERα-LBD. The concentration of ligand is held constant at 100 μM, while the concentration of ERα-LBD is decreased from top to bottom.

3420 3440 3460 3480 3500 3520 3540 3560 3580

3420 3440 3460 3480 3500 3520 3540 3560 3580

(i) (ii)

95

Figure 4.5: EPR-derived saturation binding curves for HO-2105 (diamonds) and HO-2447 (squares). Solid lines represent the best fit using a two state saturation binding model.

(c) Electronic absorption and emission properties Nitroxides are known quenchers of excited-states and have been shown to reduce

fluorescence through both inter- and intra-molecular processes 81,82. Figure 4.6 shows the

UV absorbance spectra and fluorescence emission spectra of 2 μM aqueous solutions of

E2, HO-2105 and HO-2447. The UV spectra of E2 and HO2447 both display a

characteristic absorption at 280 nm due to the phenol group that constitutes the A ring of

the steroid. The UV spectrum of HO-2105 is dominated by the diene peak at 248 nm, but

a shoulder at 280 nm is also present, confirming the presence of both diene and phenol

groups. Fluorescence spectroscopy with excitation wavelength at 280 nm shows a clear

ER-LBD concentration (μM)

0

0.00

0.0

0.01

0.0

0.02

0.0

0 5 10 15

Inte

nsity

of b

ound

com

pone

nt (a

u)

96

quenching effect of the intrinsic E2 fluorescence for the nitroxide derivatized estradiols.

We can estimate the quantum yield of HO-2105 and HO-2447 from the reported

fluorescence quantum yield from E2 of 0.11 83. The results are summarized in Table 4.2.

Figure 4.6: UV absorption spectra of E2 (solid line), HO-2105 (dash-dot line) and HO-2447 (dashed line). Right: Fluorescence emission spectra of E2 (solid line), HO-2105 (dash-dot line) and HO-2447 (dashed line).

Table 4.2: Optical parameters derived from UV and fluorescence measurements

(d) Fluorescence quenching binding assay ERα-LBD exhibits intrinsic fluorescence due to the presence of three tryptophan residues

buried within its hydrophobic core.69,84 Based on our modeling studies of the SLE docked

Ligand A280 Emax,311nm (normalized intensity)

ФF

E2 0.019 97.68 (1) 0.11 HO-2105 0.022 23.5 (0.24) 0.028 HO-2447 0.020 28.7 (0.29) 0.031

220 240 260 280 300 320 340 360 380

00.

050.

10.

150.

2

300 320 340 360 380 400 420 440 460 480 500

1020

30

4050

6070

8090

(i) (ii)

97

to available crystal structures we expect that the nitroxide moiety will be positioned well

within a 30 Å radius of the buried tryptophans, and therefore will be able to quench their

fluorescence.85 This effect can be exploited to monitor binding of the SLE. Figure 4.7

summarizes the concentration dependent quenching effect of ligand binding on the intrinsic

fluorescence of ERα-LBD, and the quenching at 340 nm is plotted vs. ligand concentration

in Figure 4.8. This curve may be fitted as previously described to provide an alternative

measure of the binding constant. Using this procedure we derived binding affinities of Kd =

7.5 μM for HO-2105 and 17.7 μM for HO-2447, in good agreement with the EPR derived

affinities. We used TEMPO to investigate non-specific quenching due to random

encounters between free spin label and excited states of the ERα-LBD. Figure 8 also shows

the non-specific quenching action of TEMPO, which exhibits significantly weaker

quenching of ERα-LBD. This observation, and the fact that the quenching effect is linear

with concentration, suggests that TEMPO does not access the protein’s interior to the same

extent as the SLEs. This result verifies that HO-2105 and HO-2447 associate with the

known estradiol binding pocket, which brings the nitroxide sufficiently close to the buried

tryptophans to efficiently quench their fluorescence.

98

Figure 4.7: Emission fluorescence of ERα-LBD as a function of HO-2105 (left) and HO-2447 (right) concentration in 50 mM Tris, 150 mM KCl, 10% glycerol, pH 8.1 at 298K.

Figure 4.8: Fluorescence-derived saturation binding curves for HO-2105 (square), HO-2447 (diamond) and TEMPO (triangle) in 50 mM Tris, 150 mM KCl, 10% glycerol, pH 8.1 at 298K.

300 320 340 360 380 400 420 440 460 480 5000

100 200 300 400 500 600 700 800 900 1000

Wavelength

300 320 340 360 380 400 420 440 460 480 5000

100

200

300

400

500

600

700

800

900

1000

Wavelength

(i) (ii)

Increase HO-2105 Increase

HO-2447

0

10

20

30

40

50

60

70

0 1 2 3 4 5

Concentration of ligand (µM)

Que

nchi

ng e

ffect

(au)

99

(e) Distance measurement of ERα-LBD homodimer using DEER DEER spectroscopy provides accurate interspin distance measurements in systems where

the two paramagnetic centers are separated by distances between 20 to 70 Å 86. ERα-LBD

is known to form homodimers in solution; in fact, the dimer form of ERα is necessary for

proper alignment of the DNA binding domain and therefore its biological action in vivo.87

We performed DEER spectroscopy on ERα-LBD bound to the SLEs as a way to both

confirm ligand binding and provide unique information about the dimer conformation as

measured from the ligand binding pocket. The DEER spectra obtained, illustrated in Figure

4.8, provide information about both distance and distance distribution between the two

paramagnetic centers in each of the receptor/ligand dimer complex. As expected from

modeling studies, HO-2105 yields a significantly shorter distance compared to HO-2447.

This is due to difference in the relative positions of the labels as well as the lengths of their

tethers. HO-2105 produces a distance of 27.5 ± 2 Å, while HO-2447 produces a mean

separation of 35.7 ± 2 Å. The uncertainty for these measurements is calculated using a

Monte-Carlo approach described in Sen et al. and the results are plotted in Figure 4.9.88

Interestingly, we observe broad distance distributions that reflect mobility of the label

within the binding pocket as well as the distribution of distances between the halves of the

ERα-LBD dimer.

100

Figure 4.9: DEER spectra and corresponding distance distribution for HO-2105 (i) and

HO-2447 (ii)

4.4 Discussion Ligand/receptor interactions critically determine the biological response of nuclear

receptors. Here we characterize a new set of biophysical tools to study ligand binding and

conformation of ERα-LBD. Attachment of a nitroxide to the native ligand of ER estradiol

transforms it into a molecular sensor capable of reporting on local dynamics and structure.

The sensitivity of this method is exemplified by the difference in lineshape features

observable between the two bound probes (cf. Figure 4.4). Lineshape analysis can

discriminate dynamic parameters that reflect details of the local environment as well as

overall binding that are characteristic for the two probes.

Binding of the SLE to ERα-LBD causes drastic changes in the observed dynamic

properties as measured by EPR. In addition to the obvious decrease in rotational diffusion

0.2 0.4 0.6 0.8 1 1.2 1.4 0

0.2

0.4

0.6

0.8

1

t (µs)2 3 4 5 6 7 8

r (nm)

0.2 0.4 0.6 0.8 1 1.2 1.4 0

0.2

0.4

0.6

0.8

1

t (µs) 2 3 4 5 6 7 8r (nm)

(i)

(ii)

101

expected from going from a freely tumbling small molecule to a much larger

receptor/ligand complex, EPR spectra are sensitive to local dynamics that result in

characteristic lineshape features. The sensitivity to local environment make EPR analysis

intrinsically more informative compared to the more commonly used fluorescence

depolarization method, where only relative mobility information can be extracted without

discrimination between local and global contributions to overall dynamics. A limitation

of the EPR method is its relatively low sensitivity compared to fluorescence and radio-

labeling methods, which limits its applicability as a competitive binding assay for high

affinity ligands. More fruitful application of the EPR binding assay method might be to

measure competitive binding of xenoestrogens.89 These steroid compounds have been

tied to human health concerns and usually display lower binding affinities to ER

compared to E2.

Nitroxides impart unique properties to the ligands in addition to EPR activity.

Distance dependent fluorescence quenching is a well known feature of nitroxides that has

recently been used to extract distance measurements in fluorescence/nitroxide labeled

systems 85. By monitoring the intrinsic fluorescence of buried tryptophans in ERα-LBD

as a function of ligand concentration, we have developed a convenient fluorescence

binding assay. Comparison of the quenching effect of SLE to that of TEMPO radical

confirms that the labeled ligands occupy the native estradiol binding pocket close to the

protein interior. Binding affinities derived from the EPR based assay and those derived

from the fluorescence quenching assay are in good agreement and therefore mutually

validating.

102

DEER spectroscopy was used to measure interspin distances between SLE/ERα-

LBD homodimer complexes. The results are consistent with crystal structures of ERα-

LBD bound to estradiol and provide further evidence that SLE bind in the expected

location of the binding pocket. In fact, crystal structures of ERα-LBD docked with HO-

2105 or HO-2447 show interspin distances comparable to the ones obtained

experimentally by DEER spectroscopy. The relatively wide distance distribution

observed can arise from a variety of contributions: (i) rotameric flexibility of the

nitroxide tether, (ii) motion of the SLE within the binding pocket, (iii) conformational

heterogeneity of the homodimer structure.

Some future applications of SLEs may be to study ligand/receptor interaction in

other estrogen-binding proteins such as ERβ or 17β-Hydroxylsteroid Dehydrogenase (17

β-HSD), as probes of local oxidative environment and as targeted contrast agents for

imaging and NMR studies90-92.

4.5 Conclusion The results presented here demonstrate that the two SLEs bind to ERα-LBD. The

nitroxide moiety imparts unique properties to the estradiol scaffold that enable

measurement of ligand binding by EPR and fluorescence spectroscopy. DEER

measurements on SLE bound to ERα-LBD homodimer provided direct structural

information on the ERα-LBD conformation. These findings indicate that this new class of

derivatized estradiols holds promise as biophysical tools for studying ligand/receptor

interaction. Work is currently underway in Prof. Hanson’s laboratory at Northeastern

103

University to explore nitroxide substitutions at the 11β position as a way to increase

binding affinity (see also Chapter 6, Future Directions).

The results here reported provide an exciting first look at a new class of

compounds with applications for xenoestrogen binding assays, structural biology of the

ER complex and ER selective imaging. This work also provides a rare example of the use

of spin-labeled ligands to probe the local binding site environment; furthermore, this is

the only example of DEER spectroscopy used to measure interspin ligand-ligand

separation. The fact that SLE may have significance in imaging of estrogen responsive

breast cancer cells should also be explored. There are at least two mechanisms that might

be employed in this case: direct detection of the unpaired electron through low field EPR

imaging or use of the SLE as ER selective relaxing agents in MRI.

Summary Chapter 3 describes the characterization of a new class of derivatized estradiols:

spin labeled estrogens (SLE). Here we describe two new methods to assay SLE binding

to ERα-LBD, EPR based binding assay and fluorescence quenching binding assay. We

also describe some applications of SLE as a biophysical probe using lineshape analysis

and DEER spectroscopy. The results reported here show the utility of these probes as

tools to investigate both ligand binding as well as solution structure of the receptor-ligand

complex.

104

Chapter 5: Other Projects

105

5.1 Development of an EPR spin trap assay to measure singlet

oxygen sensitization of DNA binding dye compounds

– in collaboration with Prof. Shana Kelley, University of Toronto, Canada

(a) Photodynamic therapy in cancer treatment The idea to use photosensitizers and light to treat disease has existed for many

years. The use of photosensitizers to treat skin cancer was initially attempted in 1903 by

the utilization of eosin and light.93 However, sensitizers such as eosin and acridine

orange are no longer used for photodynamic therapy due to their toxicity and

carcinogenicity. A rapid increase of interest in photodynamic therapy (PDT)commenced

following the first systematic clinical study of PDT for malignant lesions in 1977.93

In PDT, normally three elements are required to induce the desired result: a

sensitizer, light, and oxygen. First, the photosensitizer is administered via topical, oral,

intravascular, or local intratumor injection, followed by an incubation period that allows

the photosensitizer to disperse into the cells. Then the area of interest is irradiated with a

wavelength of light that the photosensitizer absorbs. Photoactivation of the dye results in

the sensitization of 1O2 which is reportedly the major species responsible for the ensuing

cytotoxicity.93 Photosensitizers are often taken up to a greater degree by malignant or

dysplastic tissues, which aids in their ability to cause selective cell death.94

The ideal phototherapeutic agent would have several characteristics. It would

strongly absorb light in the red region of the visible spectrum (600-900 nm), as those

wavelengths can more easily penetrate tissue. The photosensitizer would have highly

106

efficient photochemistry for killing tumor cells. The molecule would exhibit high

selectivity by localizing in tumor cells rather than healthy cells. And lastly, the drug

would rapidly clear the system following treatment, reducing unwanted side-effects.95

The Kelley laboratory developed and synthesized the photosensitizing agent here

discussed. A 1O2 sensitizing chromophore, thiazole orange (TO) was delivered into

human cells and shown to induce cytotoxicity. TO absorbs green light, as it has an

absorbance maximum at 500 nm.96 This makes it less than ideal as a phototherapeutic

agent. Therefore, it was desired to synthesize other cyanine dyes, in order to determine

whether they might be more effective at inducing phototherapy.

Carboxy functionalized cyanine dyes BO, TO, BO3 and TO3 were all synthesized

by Jay Carreon.97 These dyes could then be used for synthesizing dye-peptide conjugates

which could be examined for their ability to sensitize 1O2, enter and kill cells, and

damage biomolecules.

Much work has been done to determine how the conjugation of peptides to dyes

and other biomolecules determines where the cargo localizes in cells. Previous work

demonstrated that the TO-RrRK (R = L-arginine, r = D-arginine, K = L-lysine)

conjugates enter the nucleus of cells. Thus, it was determined to conjugate RrRK to BO,

TO and TO3 and compare their relative properties. The BO3-RrRK conjugate was not

made due to the complicated nature of the synthesis,97 and preliminary results indicating

that BO3 is a poor 1O2 sensitizer.

107

(b) 1O2 specific spin trapping using TEMP One can employ spin-trapping and electron paramagnetic resonance (EPR) to

examine the 1O2 sensitizing capability of different molecules.98-102 2,2,6,6-

tetramethylpiperidine (TEMP) has been shown to react selectively with 1O2 (as opposed

to superoxide radical) to form 2,2,6,6-tetramethylpiperidine oxide (TEMPO). In fact, if

TEMP is added to a common superoxide generating system of xanthine oxidase plus

acetaldehyde, no appreciable amount of spin-trapping is observed. Levels of 1O2 as low

as 10 nM are easily detectable.100,101 Current advanced in cavity design considerably

increase EPR sensitivity and should allow even lower levels of 1O2 to be observed.

108

N

NS

Me

OMe

O

TO-OMe

NNH

HN

O

O

O

NH

NH

HN NH2

HN

NH2

O

O

HN HN

NHNH H2NH2N

TO3-RrRK

NH2

N

NS

Me

NH

HN

O

O

O

NH

NH

HN NH2

HN

NH2

O

O

HN HN

NHNH H2NH2N

BO-RrRK

NH2

NS Me

N

NS

Me

NH

HN

O

O

O

NH

NH

HN NH2

HN

NH2

O

O

HN HN

NHNH H2NH2N

TO-RrRK

NH2

NOMe

O

TO3-OMe

NS Me

N

NS

Me

OMe

O

BO-OMe

Figure 5.1. Structures of cyanine dye methyl esters and cyanine dye-RrRK conjugates. Dyes were synthesized by Jay Carreon. Conjugates were made by Fmoc solid phase peptide synthesis.

109

EPR spin-trapping with TEMP was employed to determine whether the

methylated dyes, BO-OMe, TO-OMe, and TO3-OMe and the conjugates BO-RrRK, TO-

RrRK, and TO3-RrRK sensitize 1O2. All of the dyes sensitize 1O2 to some degree,

however TO, TO3, and conjugates of those dyes are much more efficient than BO or the

BO-conjugate tested. Interestingly, the amount of 1O2 trapped by TEMP with TO-RrRK

is similar if not greater than what is trapped when TO-OMe is irradiated, but the same

trend is not observed with TO3-RrRK conjugate and TO3-OMe. It could be that the

electron density of the chromophore is slightly redistributed by conjugation with the

peptide, or more likely that the peptide interacts with TEMP or 1O2.

DNA binding was found to influence the amount of 1O2 detected by TEMP spin-

trapping during the irradiation of TO-OMe, TO-RrRK, TO3-OMe and TO3-RrRK. Due

to the low levels of 1O2 detected in the photosensitization of BO-OMe and BO-RrRK in

the previous assay, those molecules were not tested. In general, as DNA was added to

the dye solution, a decrease in the amount of 1O2 spin-trapped also declines in a reverse

binding curve until leveling out is observed. This binding behavior becomes more

apparent when comparing the rose bengal (RB) spin trapping with the DNA binding

compounds. RB does not specifically bind DNA, which leads to a more linear decline in

1O2 trapping. The decline in TEMPO• observed in all cases suggests that 1O2 reacts with

the DNA more quickly than it can diffuse into solution to react with TEMP. There is also

a possibility that the triplet state of the cyanine dyes are influenced by the binding to the

DNA, as the photophysics are affected when the molecule is rigidified (Table 1). In

order to determine whether the triplet state is affected, one could look at the triplet-triplet

110

absorption spectra of the molecules with and without DNA. One could also look at the

fluorescence lifetime of the dyes in the presence and absence of DNA and oxygen using

single photon counting. If the relative kinetics differ, it could indicate that the

intersystem crossing rate is affected.

Figure 5.2. Photosensitization of 1O2 by dyes and dye conjugates. Dyes were irradiated in phosphate buffered saline pH 7.2 in D2O in the presence of TEMP and examined by EPR spectroscopy. The resulting signal intensity was quantitated and compared to a standard curve of TEMPO•. The background signal resulting from TEMPO present in the TEMP solution was subtracted.

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

BO-OMeTO-OMeTO3-OMeBO-RrRKTO-RrRK TO3-RrRK

[TEMPO] (1x10-6 M)

Irradiation time (min)

111

Table 5.1. Fluorescence quantum yields (Ф) for dye derivatives in buffer and DNA measured by Jay Carreon.

compound λmaxAbs

(nm)

λmaxEm

(nm)

Φrel (Buffer) Φrel (DNA)

BO-CO2Me 446 473 0.0002 0.029 ± .002c

TO-CO2Me 503 560 0.0003 0.14 ± 0.0002c TO3- 625 564 0.0012 0.024 ± 0.0003c

aMeasured in samples containing 1.5 μM dye and reported relative to a fluorescein standard. bMeasured in samples containing 1.5 µM dye and reported relative to a rose bengal standard. cMeasured in samples containing 5mM dye and 50mM CT DNA.

112

Figure 5.3. Influence of DNA binding on 1O2 sensitization as detected by spin trapping with TEMP.. To 10µM dye and 50mM TEMP increasing amounts of DNA were added. TEMP spin-trapped less 1O2 with increasing DNA concentration. The cyanine dyes show a pattern indicative of DNA binding, while rose Bengal RB does not (inset).

0

5

10

15

20

25

30

35

0 2 4 6 8 10

RB

TEMPO (1x10-6)

DNA bp/dye

-1

0

1

2

3

4

5

0 2 4 6 8 10

TO-OMeTO3-OMeTO-RrRKTO3-RrRK

[TEMPO] (1x10-6)

DNA bp/dye

113

5.2 Effect of membrane thickness on cannabinoid receptor 1

transmembrane helical conformation

– in collaboration with Dr. Elvis Tiburu and Prof. Alexandros Makriyannis, Center for

Drug Discovery (CDD), Northeastern University, Boston, MA

(a) Introduction G protein-coupled receptors (GPCRs) are cell-surface proteins characterized by

seven transmembrane helices 103-106. Cannabinoid receptors (CB1 and CB2) are GPCRs

that are activated by a variety of endogenous cannabinoid agonists (anandamide, 2-

Arachidonoylglycerol) and exogenous cannabinoids (Delta 9-tetrahydrocannabinol), and

couple to Gi/o during signal transduction.107-109 Certain key amino acid sequences in the

transmembrane helices (for example, NPXXY in TM7) define structural motifs that keep

the receptor preferentially locked in an inactive state by forming intramolecular

interactions 110-113. Agonist ligand binding activates the receptor by disrupting these

interactions, thus allowing the protein to undergo conformational transition to the active

state.110-112 The CB1 receptor is predominantly present in the central nervous system, and

is expressed in many regions of the brain including the hippocampus, cerebral cortex,

basal ganglia and cerebellum.114-116 The CB2 receptor is found in certain peripheral

tissues such as the spleen and white blood cells. There is tremendous interest in CB1

because it is among several other rhodopsin-like GPCRs that are targets for

therapeutics.117,118

Alternatively, GPCRs can also be activated through modulation of membrane

properties without any ligand interaction.110 Constitutive activation of GPCRs suggests

114

that the lipid bilayer membrane itself plays a major role in mediating mechanochemical

signal transduction, which would be expected considering the importance of lipid/protein

interactions for the function of membrane proteins.113,119,120 Chachisvilis et al

reconstituted purified GPCRs in liposomes that showed constitutive activity due to

membrane modulation in the absence of agonist ligands.113,120,121 This finding suggests

that the lipid bilayer membrane plays an active role in mediating receptor activation and

signal transduction. In this case functional domains in the transmembrane helices induce

a conformational change responsible for activation of GPCRs. Gudi et al. have also

shown that mechanical forces initiate mechanochemical signal transduction by increasing

membrane tension and altering physical properties of the cell membrane in human B2

bradykinin receptor.110,121

Utilizing synthetic peptides derived from TM7/H8 of CB1 and CB2, we have

previously demonstrated that membrane fluidity increased as a function of peptide

concentration.122 The findings also showed that the peptides increased the degree of

disordering within the bilayer differently, with CB1 influencing membrane fluidity more

than CB2 derived peptides.122 An important consideration that we explored was whether

differences in structural motif at the cytoplasmic helix 8 in both receptor peptides could

serve as potential sites for a conformational bend or twist of functional microdomains in

the peptides within the membrane, resulting in increased membrane permeability.122

These studies hypothesized that membrane fluidity was modulated by conformation

changes in TM7/H8 of CB1 and our conclusions were based on lipid headgroup

perturbation as well as changes in the acyl chain dynamics.

115

The location of certain trigger residues in close proximity in TM7/H8 supports the

notion that membrane thickness could, in addition to altering the tilt angle, also be a

driving force to modify helical conformation and structure. Here, we describe the

dynamics and conformation of the peptide fragment (TM7/H8) from the human

cannabinoid receptor 1 (hCB1) in phospholipid bilayers utilizing solid-state nuclear

magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies.

We used 2H- and 15N-labeled mechanically oriented samples of TM7/H8 to identify the

orientation within dimyristoylphosphatidylcholine (DMPC; 14:0) and 1-palmitoyl-2-

oleoyl-sn-glycero-phosphatidylcholine (POPC; 16:0, 18:1) phospholipid bilayer

environments. Solid-state NMR is an established technique used to study the orientation

of membrane proteins in lipid bilayers.123-125 Solid-state 2H NMR spectroscopy in

particular can be used to study the dynamics and orientation of side chain residues in site-

specific 2H labeled integral membrane proteins. Methyl group motions have been well

characterized by 2H NMR studies of CD3-labeled sites of Ala residues in other

transmembrane peptides.126-128 A practical advantage of using a CD3 Ala label as an

NMR probe in addition to the conventional 15N solid-state NMR spectroscopy studies is

that highly intense signals are observed due to the methyl group intrinsic mobility, and

the presence of three chemically equivalent deuterons on each side chain.129 Site specific

15N-labeled Ala studies will provide backbone dynamics and orientation whereas 2H site

specific Ala studies will reveal side chain dynamics and orientation. The chemical shifts

of singly 2H or 15N-labeled Ala TM7/H8 peptide were studied in DMPC and POPC

phospholipid bilayers mechanically oriented between thin glass plates.123,130,131

116

Site-directed spin labeling (SDSL) has emerged as a sensitive method to probe

local dynamics and conformation of proteins using EPR spectroscopy.132-135 The

nitroxide spin label used in this study is designed to covalently and selectively react with

cysteine residue forming a stable thioether bond.135-138 TM7/H8 of CB1 contains two

unique cysteine residues positioned within a complete turn of i to i+4 in TM7. We

produced singly labeled peptides to study changes in dynamics and local environment

between POPC and DMPC model membranes. Doubly labeled TM7/H8 peptide was also

used to monitor secondary structure changes via distance measurements from dipole-

dipole couplings between the two spin labels in 30% trifluoroethanol (TFE) and in the

two membrane systems. The strategy focuses on identifying structural changes of

TM7/H8 in the two model membrane (POPC and DMPC) and determining whether

structural changes are modulated by lipid bilayer environment.

(b) Experimental Procedures POPC and DMPC in chloroform solutions were purchased from Avanti Polar

Lipids (Alabaster, AL) and stored at –20 °C prior to use. 2,2,2-Trifluoroethanol (TFE),

N-[2-hydroxyethylpiperazine-N’-2-ethane] sulfonic acid (HEPES) and EDTA were

obtained from Sigma-Aldrich (Milwaukee, WI). Deuterium-depleted water was

purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were purchased

from Sigma-Aldrich (St. Louis, MO). The spin label employed in this study is [3-

(iodomethyl)-2,2,5,5-tetramethyl-2,5-dihydropyrrol-1-oxyl], and was purchased from

Toronto Research Chemicals (Toronto, ON).

117

Peptide synthesis and purification: Peptide corresponding to TM7/H8 [hCB1(T377-

E416)] was synthesized using an Applied Biosystems peptide synthesizer (ABI 433A)

equipped for FMOC chemistry at the Molecular Biology Core Facility, Genescript Inc.

The numbering system suggested by Bramblett and coworkers was employed.139 All

amino acids were single coupled, with a total of 9.5 min for the coupling and monitoring

module. The crude peptide was purified on a Dynamax Model, SD-200 high performance

liquid chromatography system, controlled by an Applied Biosystem 1000S Diode Array

Detector. The software used was Dynamax Method Manager (version 1.4.6). The purified

peptide was analyzed by MALDI-TOF using a calibration matrix of α-cyano-4-

hydroxycinnamic acid.

NMR and EPR sample preparation: Bilayers of DMPC and POPC were prepared with a

2 mol% ratio of peptide to lipid according to a slightly modified protocol, as

described.140-143 Appropriate standard solutions of either DMPC or POPC phospholipids

in chloroform were mixed with a suitable amount of peptide dissolved in TFE to give the

desired molar composition (1:100 peptide to lipid molar ratio).142,143 The solvents were

removed under a steady stream of N2 gas in a 12 x 75 mm test tube at ambient

temperature144 and the samples placed in a vacuum desiccator overnight. The 15N NMR

powder sample was then transferred and packed into a 4 mm ZrO2 rotor. The hydrated

sample was prepared by placing the rotor containing the dry sample in a sealed chamber

with saturated ammonium monophosphate (relative humidity 93%). The sample was then

incubated for 6-12 hrs at 45 °C.

118

The mechanically aligned sample was prepared by dissolving the peptides in a

minimal amount of TFE and the solution mixed with either DMPC or POPC (1:100

peptide to lipid mole ratio was used) dissolved in chloroform (20mg/mL) in a pear-

shaped flask. Nitrogen gas was passed through the resulting mixture to reduce the volume

of chloroform to a third of the original volume. The sample was spread on 25 glass plates

of 8.5 mm x 14 mm dimensions and was allowed to dry in a desiccator overnight.145

Deuterium-depleted water was added onto the lipid-peptide mixture and the glass plates

were stacked on top of each other. The stacked glass plates were then placed in a

humidified chamber of ammonium monophosphate at a relative humidity of ~ 93% at 42

°C until the sample became transparent, indicating complete incorporation of the peptide

into the membrane.

Site directed spin labeling was achieved by resuspending the lyophilized peptide

in a solution of 4 M guanidine-HCl, and adding appropriate amount of 0.1 M stock spin

label solution for a final ratio of 3:1 spin label to peptide. The mixture was allowed to

react overnight at 4oC before being purified and characterized. Extra spin label was

removed by dialysis using a 500 Da cut-off membrane. Reverse phase HPLC was further

used to separate the peptide from the unreacted label. Fractions were analyzed by MS to

confirm labeling and by EPR to confirm the nitroxide’s activity.

NMR spectroscopy: For the 2H NMR studies, the quadrupolar echo pulse sequence was

used with quadrature detection capabilities with complete phase cycling of the pulse

pairs. A 3.0 μs 900 pulse, a sweep width of 100 kHz, a recycle delay time of 400 ms, and

a 30 μs interpulse delay were used to accumulate 150,000 transients. Prior to Fourier

119

transformation, an exponential multiplication of 200 Hz line broadening was performed

on the spectra. 2H NMR spectra were obtained at 35 °C for the mechanically aligned

TM7/H8 samples.

15N-labeled solid-state samples were placed in a 4 mm sample rotor in a Bruker

AVANCE 500 WB solid-state NMR spectrometer operating at 50.7 MHz for 15N and

equipped with a triple resonance CPMAS solid-state NMR probe. 15N solid-state NMR

spectra were collected utilizing a standard cross polarization pulse sequence with 1H

decoupling. The following pulse sequence parameters were used; 4.7 μs 1H 90°, 500 ppm

sweep width, 1.5 ms contact time and a 4 s recycle delay with 1H decoupling 144,146. The

spectra were referenced to an external standard of (15NH4)2SO4, (27 ppm). The

experiments were recorded at 35 °C for both DMPC and POPC samples.

For the 2H and 15N-labeled mechanically aligned samples, a double resonance flat coil

probe was used operating at the required temperature in a 700 MHz spectrometer. For the

15N-labeled samples, the following pulse sequence parameters were used: 4.5 μs 1H 90°

pulse, 1.0 ms contact time, 600 ppm sweep width, and a 4 s recycle delay with 1H.129,147-

149

EPR spectroscopy: EPR measurements were conducted on a Bruker EMX instrument

equipped with high-sensitivity cylindrical cavity and fitted with variable temperature

module. For aqueous samples at 308 to 353 K, spectra were was acquired at a microwave

frequency of 9.37 GHz, 2.0 mW microwave power, with 0.5 G 100 kHz field modulation

amplitude. Rigid limit spectra were acquired at 150 K with, 0.2 mW microwave power,

and 1.0 G modulation amplitude, with all other conditions kept the same.

120

(c) Results According to modeling studies, Ala380 is believed to be part of TM7 and

is close to the phospholipid bilayer/water interface.139 The numbering system corresponds

to the position of the amino acids in the full length wild-type receptor proposed by

Bramblett et al.139 We conducted 2H NMR studies of site-specific CD3 labeled Ala380

TM7/H8 peptide mechanically aligned with the bilayer normal perpendicular with respect

to the static magnetic field. The rotation about the Cα-Cβ can be characterized as three

site hopping with a quadrupolar splitting of about 40 kHz for unoriented samples. The 2H

quadrupolar splitting of the unoriented samples of POPC spectrum of Ala380 (Figure 5.4

A) is 40 kHz indicating a peptide with restricted side chain mobility. Similar quadrupolar

splitting has been observed in randomly dispersed 2H-labeled Ala samples of unoriented

Ala residues of other TM proteins incorporated into POPC bilayers. In the aligned POPC

spectrum, the quadrupolar splitting of Ala38 was 32.5 ± 0.1 kHz (Fig. 5.4 B) and 31.0 ±

0.1 kHz in DMPC. The variance was estimated from three different trials. The decreased

quadrupolar splittings of Ala in going from the unoriented to the aligned spectra is most

likely due to an increased mobility of the Ala380 residue. The resolved Pake doublets

clearly show that the sample is well aligned in POPC and DMPC when compared to the

powder spectrum, indicating the peptide is embedded within the lipid bilayer (Fig 5.4 A).

In addition to the 31 kHz Pake doublet observed in the DMPC spectra there are additional

Pake doublets with quadrupolar splittings of 10.3 and 14.1 kHz. These additional Pake

doublets are not observed in the POPC sample. We attribute these inner Pake doublets to

conformational heterogeneity of the peptide in the DMPC membrane environment.

121

We conducted solid-state NMR experiments on 15N-labeled Ala380 in TM7/H8

mechanically aligned on glass plates. 15N solid-state NMR spectroscopy of oriented

membrane peptides has been useful for investigating the polypeptide backbone secondary

structure and topology.140 The spectrum in Figure 5.4 D was obtained from an unoriented

sample of the 15N-labeled Ala380 in TM7/H8 peptide in POPC phospholipid bilayers. In

Figure 5.4 D we noted that the unoriented spectra spans from 215 to 70 ppm indicating

that TM7/H8 backbone is quite rigid in the NMR time scale within the membrane. The

spectrum in Figure 5.4 E was obtained from an oriented sample of the 15N-labeled

Ala380 in TM7/H8 peptide in POPC phospholipid bilayers mechanically aligned on glass

plates. There is a relatively sharp resonance peak at 200 ppm due to the 15N-labeled

Ala380 residues in TM7/H8. Figure 5.4 F was obtained from the same labeled peptide in

aligned DMPC phospholipid bilayers, and exhibits a broad resonance with chemical shift

at 180 ppm. The 15N–labeled Ala380 resonance peaks in POPC and DMPC are downfield

of the unoriented 15N sample (Figure 5.4 D) indicating the residue is embedded within the

phospholipid bilayer. The change in chemical shift between POPC and DMPC reflects a

change in peptide orientation within the two bilayers. However, we also observe an

envelope of spectral overlap of the DMPC sample with intrinsic broadness which

supports the 2H aligned spectrum shown in Fig. 5.4 A and indicates a peptide with

conformational heterogeneity. In order to probe the local conformational properties we

conducted site directed spin labeling and EPR studies on the peptide reconstituted in

DMPC and POPC bilayers.

122

Figure 5.4: One-dimensional solid-state 2H and 15N NMR spectra of site-specific 2H or 15N-labeled Ala380 TM7/H8 in oriented DMPC and POPC phospholipid bilayers. The spectra displayed were collected at 308K for both DMPC and POPC phospholipid bilayers. (A) Chemical shift tensor of unoriented 2H-labeled Ala380 TM7/H8 in POPC, (B) 2H-labeled Ala380 in POPC, and (C) 2H-labeled Ala380 in DMPC. (D) Chemical shift tensor of unoriented selectively 15N-labeled TM7/H8 in POPC, (E) 15N-labeled Ala380 in POPC, and (F) 15N-labeled Ala380 in DMPC. The sequence representing the amino acid residues of helix 7 and the cytoplasmic helix 8 of the cannabinoid receptor-1 receptor with 15N-labeled Ala380 shown in bold face.

Investigation of the dynamic properties of the polypeptide within the

membranes was conducted by SDSL-EPR of peptides singly labeled near the

phospholipid bilayer/water interface at Cys382 (Figure 5.5). Doubly labeled peptides

were used to determine dipolar interactions between Cys382 and Cys386 (i to i + 4) from

which the distance between the two spin labels was estimated using a dipolar broadening

deconvolution method.150 Detailed information about the local dynamics of spin labeled

polypeptides may be obtained by least-squares analysis of the EPR lineshape of singly

Dm hCB1(T377-E416) - TVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPSCE

123

labeled peptides.151 In nitroxide spin labeled systems both overall molecular tumbling and

local motion of the label affect the EPR lineshape. Figure 5.6 shows the EPR spectra of

TM7/H8 labeled at Cys382 and reconstituted in POPC, DMPC and TFE/H2O mixture,

DMPC at 308 K. The spectral lineshapes observed in TFE/H2O mixture and POPC are

quite similar, indicating that the nitroxide is experiencing similar structural environments

consistent with fast tumbling of the polypeptide and reorientation of the flexible nitroxide

tether. In DMPC, the nitroxide spectrum shows remarkably different characteristics

including broadening of the resonance lines and lineshape features suggestive of a

comparatively less mobile nitroxide tether.

124

Dm hCB1(T377-E416) - TVFAFCSMLALLNSTVNPIIYALRSKDLRHAFRSMFPSAE

Figure 5.5: EPR spectra of spin labeled TM7/H8 reconstituted in POPC, DMPC and TFE/H2O mixture at 308K. Dashed lines show the results of least squares fitting calculated based on the parameters in Table 1. The sequence representing the amino acid residues of helix 7 and the cytoplasmic helix 8 of the cannabinoid receptor-1 receptor is shown at the top with Cys382 shown in bold face.

We used non-linear least-square lineshape analysis to estimate dynamic

parameters for the three cases under study. As summarized in Table 5.1, the rotational

diffusion parameters (R║ and R┴, with respect to the nitroxide molecular frame), isotropic

hyperfine spitting (Aiso) and mobility parameter ΔH0−1 were estimated for polypeptides

reconstituted in TFE/H2O mixture, POPC, and DMPC using two site fittings. The two site

fitting was chosen based on spectral features (e.g. peak asymmetry, shoulder features)

which suggest the presence of multiple components. Both the rotational parameters and

the more qualitative ΔH0−1 mobility parameter are consistent with the observation that the

0.2 0.4 0.6 0.8 1 1.2 1.40

0.2

0.4

0.6

0.8

1

t (µs)

Nor

m. I

nten

sity

2 3 4 5 6 7 8r (nm)

0.2 0.4 0.6 0.8 1 1.2 1.40

0.2

0.4

0.6

0.8

1

t (µs)

Nor

m. I

nten

sity

2 3 4 5 6 7 8r (nm)

-400 -300 -200 -100 0 100 200 300 400

DMPC

TFE

POPC

Field (Gauss)

125

nitroxide is experiencing similar dynamic environments in TFE/H2O mixture and POPC,

while in DMPC the observed mobility is considerably reduced. In TFE/H2O mixture the

two sites have equivalent Aiso as expected from the presence of a single hydrophilic

phase, whereas peptides reconstituted in POPC and DMPC membranes show the

presence of two phases, with the POPC samples experiencing marked differences

between the two environments.

Table 5.1: Summary of rotational diffusion parameters R║ and R┴ expressed in log10(s-1), isotropic hyperfine spitting, Aiso in MHz, the percent contribution, and mobility parameter ΔH0

−1 in gauss (G) calculated from non-linear least squares fits of spin labeled peptide in POPC, TFE/H2O mixture, and DMPC at 308 K. Fixed parameters in the fits include g-factor (gx, gy, gz) = (2.0085, 2.0056, 2.0020) and inhomogeneous Gaussian line width of 1 G.

POPC TFE/H2O DMPC Site 1 Site 2 Site 1 Site 2 Site 1 Site 2

R║ (log10) 9.13 8.25 9.13 8.19 8.0 7.5 R┴ (log10) 8.88 8.11 8.98 8.12 8.0 7.7 Aiso (MHz) 45.1 43.0 45.10 45.1 43.7 43.0 % pop. 74% 26% 15% 85% 34% 66% ΔH0

−1 (G) 0.73 0.68 0.3

The parameters calculated for spin labeled TM7/H8 in TFE/H2O mixture are

consistent with the fact that the nitroxide is experiencing a low viscosity hydrophilic

solvent in contrast with the POPC samples which show both hydrophilic and hydrophobic

environments. The hydrophilic environment experienced by the nitroxide spin label in

POPC is probably due to its vicinity to the phospholipid headgroup/water interface

region. The smaller Aiso values derived from the DMPC sample implies that the nitroxide

126

is embedded within the phospholipid bilayer in this case, which also affects both the

backbone dynamics and internal rotameric freedom of the spin label.

Nitroxide spin labeled at position Cys382 in TM7/H8 was reconstituted in POPC

phospholipid bilayers to evaluate the effects of temperature on the dynamics of the

nitroxide spin label. As shown in Figure 5.6, the temperature-dependent EPR spectra

indicate two components for this sample in the temperature range from 318 to 343 K,

which are particularly visible at higher temperatures. We used two sites fitting to

estimate Aiso and extract the relative contributions of each component to the spectral

intensity [49]. The results of the fits are summarized in Table 5.2 and confirm the

presence of a hydrophilic phase with Aiso of 45.1 MHz (site 1 in Table 5.2) and a

hydrophobic phase with Aiso of 43.0 MHz (site 2 in Table 5.2). This data suggests that the

local environment surrounding the nitroxide spin label on the peptide is distributed

between the phospholipid/water interface and the hydrophobic core of the POPC lipids.

At lower temperature the hydrophilic phase component accounts for 74% of the spectral

intensity. As the temperature is increases, so does the fraction of the hydrophobic

component which makes up 85% of the spectral intensity at 343K. This behavior is

reversible upon cooling back to 308 K. The TFE/H2O mixture sample shows minor

changes that can be attributed to increased mobility and lower viscosity at higher

temperatures (see Figure 5.7 A). In contrast, the same experiments on the peptides

reconstituted in DMPC lipids show significant increase in spin label mobility at higher

temperatures (Figure 5.7 B).

127

The equilibrium constant for the temperature dependent hydrophilic/hydrophobic

partitioning in TM7/H8/POPC has been estimated from the relative populations

calculated in the least-squares fits. Figure 5.8 shows a van’t Hoff plot from which the

changes in enthalpy (ΔH = +62 kJ/mol) and entropy (TΔS = +60 kJ/mol) may be

determined for the transition from the hydrophobic to the hydrophilic environment. The

signs of Δi and Δi are consistent with a thermally driven immersion of the polar label into

the membrane interior, so that ΔH reflects the energy cost of forcing the polar probe into

the hydrophobic environment [50, 51].

Dm hCB1(T377-E416)– TVFAFCSMLALLNSTVNPIIYALRSKDLRHAFRSMFPSAE

Figure 5.6: EPR spectra of TM7/H8 singly labeled at Cys382 reconstituted in POPC at temperatures from 318 K to 343 K. Dashed lines show the results of two site least square fitting: for the hydrophilic site Aiso = 45.1 MHz, while for the hydrophobic site Aiso = 43.0 MHz. Fixed parameters in the fits include g-factor (gx, gy, gz) = (2.0085, 2.0056, 2.0020) and inhomogeneous Gaussian line width of 1 Gauss. The sequence representing the amino acid residues of helix 7 and the cytoplasmic helix 8 of the cannabinoid receptor-1 receptor is shown at the top with Cys382 shown in bold face.

-300 -

200 -100

0 100

200

300

400

318 K

323 K

328 K

333 K

343 K

Field (Gauss)

128

Table 5.2: Fitting parameters derived from two site least square fits of singly labeled TM7/H8 peptide reconstituted in POPC at different temperatures. Site 1 represents the hydrophilic component with a fixed Aiso of 45.1 MHz and (gx, gy, gz) = (2.0085, 2.0056, 2.0020). Site 2 describes the hydrophobic component with fixed Aiso of 43.0 MHz and (gx, gy, gz) = (2.0085, 2.0056, 2.0020). R || and R┴ correspond to the parallel and perpendicular rotational correlation times and are expressed in units of log10(s-1).

log10 R || log10 R ┴ Population site 1 (%)

Temperature (K)

site 1 site 2 site 1 site 2 308 9.13 8.25 8.88 8.11 74 323 9.25 8.25 8.64 8.5 38 328 9.33 8.28 8.55 8.75 35 333 9.5 8.35 8.57 8.89 21 343 9.45 8.53 8.6 8.88 15

-600 -400 -200 0 200 400 600 -600 -400 -200 0 200 400 600

Figure 5.8: EPR spectra of TM7/H8 singly labeled at Cys382 reconstituted in TFE/H2O mixture at 308 and 343 K (A) and in DMPC at 308 and 343 K (B).

A B

343 K 343 K

308 K308 K

Field (Gauss) Field (Gauss)

129

0.0028 0.0029 0.003 0.0031 0.0032 0.0033 0.0034-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

1/T

Ln(K

)

Figure 5.7: Van’t Hoff plot derived from relative populations of hydrophobic/hydrophilic components as a function of temperature. The equilibrium constant K at temperature T was computed by taking the ratio of relative populations of hydrophobic to hydrophilic components derived from fits. The best fit line ( ln(K) = −7542/T + 23.8 ) provides the estimates ΔH = +62.7 kJ/mol and TΔS = +60 kJ/mol for the transition from the hydrophobic to the hydrophilic environment.

Doubly labeled TM7/H8 at positions Cys382 and Cys386 (i and i+4) was

prepared and reconstituted in TFE/H2O mixture, POPC, and DMPC. The spin-spin

dipolar interaction in doubly labeled systems can be used to extract accurate interspin

separations in the range of 5 to 20 Å from the CW-EPR spectrum 152. Figure 5.8 shows

the rigid limit EPR spectra measured for doubly labeled TM7/H8 peptide in TFE/H2O

mixture, POPC and DMPC lipids. The spectral reconstruction method developed by Fajer

et al. 152 allows evaluation of both the amount of broadening caused by dipolar

interaction as well as the percentage of non-interacting component caused by incomplete

130

labeling or misfolding of the labeled peptide leading to interspin separations beyond the

20 Å detection limit 150. Table 5.3 summarizes the results of the fits. Peptides

reconstituted in TFE/H2O mixture and POPC have a significantly higher fraction of

coupled spins than the DMPC reconstituted peptide. Since all the samples studied were

prepared from the same stock of labeled peptide, the drastic reduction in dipolar coupling

is most likely attributable to conformational heterogeneity of the peptide in DMPC that

brings the paramagnetic centers out of the 20 Å range observable by the dipolar

broadening technique. Distances extracted from electron spin dipolar broadening are

consistent with distances measured between the same two Cys in preliminary high-

resolution NMR measurements recently obtained in Prof. Makryiannis’ lab.

Table 5.3: Results of the dipolar deconvolution for the three cases examined. The error was estimated using a 90% tolerance around the best χ2 value.

Sample Average Distance Distribution % Coupling

TFE/H2O 10.6 ±1.5 Å 8 ±3.5 Å 50 ±1.7 POPC 16.8 ±0.5 Å 6.2 ±3.5 Å 60.3 ±3.5 DMPC 11.6 ±2 Å 6.2 ±5 Å 14.3 ±1

131

Sm hCB1(T377-E416)– TVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPSAE

Figure 5.9: Rigid limit spectra of doubly labeled TM7/H8 in TFE/H2O MIXTURE, POPC and DMPC. All spectra were signal averaged for 30 scans. The sequence representing the amino acid residues of helix 7 and the cytoplasmic helix 8 of the cannabinoid receptor-1 receptor is shown at the top with Cys328 and Cys386 shown in bold face. Starting from the top, each set of plots consists of single labeled reference spectrum, double labeled spectrum, calculated fit and residual.

(d) Discussion The lipid bilayer is a dynamic structure held together by hydrophobic and lipid/lipid

interactions 153. Because of the high degree of spatial inhomogeneity, the interior of the

lipid bilayer is characterized by strong lateral pressure forces that vary greatly with the

depth within the bilayer 153. It has been suggested that changes in bilayer pressure

profiles are responsible for activation of certain membrane proteins such as gated

membrane channels and GPCRs 153. The crystal structure of rhodopsin has revealed

important structural features concerning all GPCRs. In the rhodopsin structure, the

TFE POPC DMPC

132

orientation of TM7 within the bilayer is dictated by a conformational change in H8. This

conformational change is governed either by the surrounding environment or the

interaction of the polar residues with the lipid headgroups.

We have used a combined NMR and EPR approach to determine the effect of

membrane thickness on the conformation of TM7/H8 peptide representing the C-terminus

of the cannabinoid receptor 1 in two model membranes. The differences in the 2H and 15N

NMR spectra produced in POPC and DMPC lipids suggest that the peptide’s structural

ordering is affected by the shorter DMPC membrane. First the 2H quadrupolar splittings

of Ala380 labeled TM7/H8 in both POPC (33 kHz) and DMPC (31 kHz) phospholipid

bilayers were slightly different within the limits experimental error (± 0.1). The

differences in splitting of the labeled sites in the two different phospholipid bilayers as

well as the appearance of several Pake doublets in the DMPC sample may be due to

several reasons: temperature effects, differences in protein-lipid interactions, a decrease

in wobbling of the side chain Ala380 peptide in POPC lipids relative to DMPC, a change

in the tilt of the TM7/H8 peptide, or a combination of all these different effects. First of

all, the temperature did not have any major effect on the 2H quadrupolar splittings and

15N chemical shifts shown in Figure 1 because we conducted the same experiments at

lower temperatures. The same 2H Pake patterns and 15N chemical shifts were obtained at

both 25oC and 35oC for the mechanically aligned samples. Thus the differences in the

spectra in POPC and DMPC could be due to multiple effects associated with all the

factors mentioned above.

133

To further investigate these differences we applied site directed spin labeling and

EPR as a more sensitive probe of the local environment. EPR measurement of peptide

labeled at position Cys382 confirms marked differences in the local dynamics of the

peptide reconstituted in TFE/H2O mixture, POPC and DMPC. TM7/H8 produces similar

fast motional spectral lineshapes in POPC and TFE/H2O mixture, in contrast with the

more complex slow motional spectrum observed in DMPC. A temperature study of the

TM7/H8 reconstituted in POPC lipids over the range of 308 to 343 K showed the

presence of two components in the EPR spectrum that can be attributed to a hydrophilic

phase that predominates at lower temperatures, and a hydrophobic phase that becomes

more pronounced at high temperature. Partitioning between hydrophobic and hydrophilic

environments suggests that the nitroxide is positioned near the phospholipid bilayer/water

interface. As the temperature is increased, the peptides undergo conformational changes

that position the label in a more hydrophobic environment. This interpretation is

supported by ΔH and ΔS values extracted from van’t Hoff analysis of the relative

populations. In contrast, the peptide does not undergo the same temperature-dependent

partitioning in DMPC, demonstrating different orientation and dynamics of the nitroxide

reporter group within the membrane.

We measured dipolar broadening in doubly labeled peptide reconstituted in

TFE/H2O mixture, POPC and DMPC lipids as a way to extract interspin distances. The

results highlight the effect of hydrophobic mismatch on the structure of the peptide.

Samples reconstituted in TFE/H2O mixture and POPC showed a high degree of coupling

with distances of 10.6 (± 0.5) Å and 16.8 (± 1) Å respectively, whereas the DMPC

134

reconstituted peptide showed a much lower level of interspin interaction. The large

coupling between labels located at i and i+4 is strongly indicative of an α-helical

structure in TFE/H2O mixture or POPC, whereas the peptide undergoes a conformational

adjustment in the shorter DMPC bicelles that causes the interspin distances to exceed the

20 Å detection limit of the dipolar interaction technique. The average distance between i

and i+4 labels of the same peptide in TFE/H2O mixture was estimated from the high-

resolution NMR structure to be 7 Å. The interspin distance measurements from the EPR

compare with the high-resolution structure considering the tether length and rotamer

structure of the spin labels.

In Figure 5.10 we present a molecular model of Cys382 spin labeled TM7/H8 to

help visualize the position of the nitroxide spin label within the POPC lipid bilayer. The

16.8 Å interspin distance between Cys382 and Cys386 (i to i+4) residues measured in

TM7/H8 indicates an extended helix in POPC which justifies positioning the nitroxide

reporter group close to the headgroup/water interface. As the temperature is elevated the

probe more frequently samples the hydrophobic environment due to increased peptide

motion and membrane phase transitions. In DMPC, the majority of the interspin distances

observed for TM7/H8 are outside the range of the dipolar-broadening technique applied

here. However, the measured distance of 11.6 Å, which corresponds to only 14% of the

peptide population, places the spin probe well within the hydrophobic bilayer.

Conformational heterogeneity in DMPC underlines the contrast with the POPC case

where more than 60% of the peptide population produces dipolar coupling. We take this

135

as evidence that modulation of the bilayer thickness has a direct effect on the

transmembrane helix conformation.

Figure 5.10: Molecular model of TM7/H8 in POPC with the nitroxide label at position 382 depicted in blue. The model shows the proximity of the nitroxide reporter group to the lipid headgroup/water interface.

When we combine the solid state NMR and EPR results the emerging picture

provides insights into the structure and dynamics of the membrane bound TM7/H8

peptide. In the POPC lipid environment, there exists essentially one conformation for the

peptide, whereas DMPC induces conformational heterogeneity in the peptide. The NMR

data therefore show that reducing the membrane thickness decreases the conformational

ordering of the peptide within DMPC. Spin label experiments confirm this result and add

structural details about the environment of the spin probe.

136

Implication of membrane hydrophobic thickness for backbone flexibility of TM7/H8.

TM7/H8 is an integral domain of CB1 that is strongly tied to receptor activity. In

this context, the results presented here have direct implications for the biological function

of the CB1 receptor. Constitutive activation drives compartment-selective endocytosis of

CB1 across membranes of different thickness 154. Structural remodeling across

membranes requires conformational modulation of the transmembrane helices. A key

feature of the TM7/H8 ability to respond to different membrane thicknesses depends on

the presence of a conserved motif NPXXY which is present in most GPCRs. Proline

residues are known to destabilize α-helices in globular proteins, but in transmembrane

helices such as those of the cannabinoid receptors, the NPXXY motif may provide a

flexible α helix which is sensitive to hydrophobic mismatch and a response mechanism

that allows conformational modifications when moving from one membrane system to

another. Characterization of the NPXXY motif in various single- or multi-transmembrane

spanning proteins also reveals its diverse role in mediating internalization and targeting

120,154. Normally long and rigid transmembrane domains compensate for hydrophobic

mismatch when reconstituted into shorter membranes by tilting. However, for flexible

helices with NPXXY microdomains, other mechanism such as kinking and

conformational changes might be important. We postulate that the kink at P394 residue in

TM7 is crucial for conformational modulation in DMPC, which may occur in concert

with additional structural rearrangements in the other CB1 receptor transmembrane

helices, and may be important for constitutive activation of GPCRs.

137

(e) Conclusion In conclusion, our solid-state NMR and EPR data on the structure of the 40-mer peptide

in POPC clearly indicate that the first 23 amino acid residues form an α-helical structure

embedded within the bilayer. This description is supported by a recently acquired high-

resolution structure of TM7/H8 in detergent micelles (unpublished experiments). When

the same peptide is introduced into the narrower DMPC bilayer, a distinct change in the

structure of the transmembrane segment is observed, which suggest that the peptide has

variable structural elements within DMPC lipids. Our results thus suggest that the bilayer

width can play a role in modulating the structure of the transmembrane helices within

GPCRs. The observed change in conformation of the TM7/H8 may be in response to

membrane thickness and provides important evidence for the molecular mechanism of

constitutive endocytic activity observed in most GPCRs. This might be evidence that

compartment-selective endocytosis of CB1 across membranes of different thickness

requires a conformational change within the transmembrane helix.

138

5.3 Molecular-scale force measurements in a coiled-coil peptide by electron spin resonance – in collaboration with Guarav Sharma and Prof. Dinos Mavroidis, Department of Mechanical Engineering, Northeastern University, Boston, MA

Fabrication of multi-component nanostructures requires the assembly of

molecular scale components into ordered arrays. Biological systems offer examples of

self-assembling structures that come together to form functional entities 155. Coiled-coil

peptides are particularly interesting biological models that naturally form robust

multimers, and that can be tuned to yield dimers and trimers, as well as large fiber

assemblies with predictable morphologies 156-160 . In order for these structures to find

applications as nanodevices, new methods are being developed that predict and measure

their mechanical properties at the nanoscale level 161-163. In this section we demonstrate a

new experimental method for measuring inter-coil forces that is based on electron spin-

labeling and double electron-electron resonance (DEER) spectroscopy.

The model system used for these measurements is based on the α-helical coiled

coil leucine zipper portion of the yeast transcriptional activator GCN4 164 (residues 249-

281 of PDB entry 1YSA; hereinafter GCN4-LZ). The coiled-coil structure consists of

two identical polypeptide chains ~4.5 nm long and ~3 nm wide.

To accomplish the force measurement, a leucine zipper peptide was synthesized

with a spin label attached at residue 246 near the N-terminus, as shown in Figure 5.11.

This location places a label near the end of the each helix, with the objective of forming

the coiled-coil dimer and determining the distribution of distances between the two labels

as described below. The Multicoil score 165 was used to estimate the propensity of this

sequence to form a coiled-coil leucine zipper, based on the identities of the amino acids

139

in positions a-g of the coil (cf. Figure 5.11), where a score between 0.5 and 1.0 indicates

probable coiled-coil formation. Neglecting the TOAC residue, the score for the GCN4-

LZ sequence is 0.83, indicating a strong propensity to form a coiled-coil dimer that is

most likely reinforced by the tendency of the TOAC residue to adapt a helical backbone

conformation.

Distance measurements can be obtained by measuring the electron spin-spin

dipolar interaction using double electron-electron resonance (DEER) spectroscopy.166

The TOAC spin label 167-169 was selected for this application because of its rigid fused

ring structure (shown in Figure 5.11), which eliminates motion of the spin-bearing

nitroxide group relative to the peptide backbone, thus ensuring that spin-spin distance

measurements directly reflect the inter-backbone distance. The GCN4-LZ sequence was

constructed with a triglycine sequence added N-terminal to the TOAC added to further

reduce the mobility of the label 167,168,170,171.

140

Figure 5.11. Coiled-coil leucine zipper structure investigated in this work, indicating structure and position of the TOAC spin label in the sequence and within the leucine zipper motif. Right: cartoon of the coiled-coil dimer conformation, showing the location TOAC labels.

Solid phase peptide synthesis using Fmoc-protection chemistry was performed by

the research group of Prof. Gary Lorigan of Miami University (Oxford, OH) on a 433A

Peptide Synthesizer from Applied Biosystems Inc., Foster City, CA.

Figure 5.12 shows a CW-EPR spectrum from the TOAC-labeled coiled-coil

structure at pH 7. Dynamic parameters (cf. Fig. 5.12 caption) were obtained by least-

squares fitting of the slow-motional lineshape151,172 with consensus magnetic parameters

for TOAC.170,173 The labeled coil-coil structure exhibited significantly anisotropic

motion, with the principal diffusion axis (i.e. the long axis of the dimer) perpendicular to

a

a'

b

b'c

c'

d

d'

e

e'

f f'

g

g'

abcdefg abcdefg abcdefg abcdefg WT GGGXRMKQLEDK VEELLSK NYHLENE VARLKKLVGER

ONH

ON

TOAC

141

the magnetic x direction and approximately 30° away from magnetic z, consistent with

the direction estimated from a molecular model. Also shown in Figure 5.12 is the CD

spectrum of the peptide confirming the Multiscore prediction of a coiled-coil

conformation.

Figure 5.12. X-Band CW-EPR spectrum of the labeled coiled-coil dimer at room temperature. Dotted line shows least-squares slow-motional lineshape. Least-squares values of the rotational diffusion constants parallel and perpendicular to the major axis of the molecule (shown by dashed line) were R|| = 1.8×109 s−1, R⊥, = 1.9×107 s−1, with diffusion tilt angle βD= 25°. Fixed-value parameters in the fit were the electronic g-factor (gx, gy, gz) = (2.0085, 2.0056, 2.0020), 14N hyperfine tensor (Ax, Ay , Az) = (21, 9.0, 105) MHz, inhomogeneous Gaussian linewidth 1.9 Gauss, and diffusion tilt angle αD = 90°. Inset: CD spectrum of the peptide displaying the characteristic features of an alpha-helical coiled-coil.

3460 3480 3500 3520 3540 3560 3580Magnetic Field (Gauss)

200 220 240-

0

1

2

3

Wavelength

Elli

ptic

ity βD

142

Four-pulse double electron-electron resonance (DEER) spectroscopy was

performed at 65 K, and the results are summarized in Figure 5.13. The time domain

signal (Figure 5.13 A) was baseline-subtracted and then Fourier-transformed to give the

spectrum shown in Figure 3B. The spectra reflect a frequency distribution given by

( )3

22210

41cos3

hrgg

πθβμν −

= [5.1]

where g1 and g2 are the isotropic g-factors of each electron, β is the Bohr magneton, μ0 is

vacuum permeability, h is Planck’s constant, r is the interspin distance, and θ is the angle

between the magnetic field and the interspin vector. The spectrum exhibits the

characteristic “Pake pattern” with turning points corresponding to θ = 0° and θ = 90° (cf.

Eq [5.1]) from which the distribution of interspin distances r can be derived. Figure 5.13

C shows the distance distribution functions P(r) obtained from the DEERAnalysis2006

program 174 using model-independent Tikhonov analysis. The resulting distribution of

distances exhibited a large fraction of the population at a distance of 2.2 nm, very close to

the distance of 2.3 estimated from a molecular model of the TOAC-labeled dimer

conformation (Fig. 5.11). The model-independent analysis also indicated a small fraction

of spins with larger separation distances, which may reflect a minor degree of interaction

between coiled-coil units. The distribution of distances in the main fraction is very

narrow (about 0.14 nm), confirming that the coiled-coil adopts a compact and well-

defined structure.

143

0 0.5 1Time (μ sec)

A

-20 0 20Frequency (MHz)

B

1.5 2 2.5 3Distance (nm)

C

Figure 5.13: (A) Time domain DEER signal showing modulation from spin-spin interaction. (B) Frequency domain DEER signal showing characteristic Pake pattern of an isotropically distributed pair of dipoles, obtained by Fourier transform of the data in (A) after baseline subtraction (straight line); (C) Solid line shows distribution of distances between spin labels obtained by model-independent Tikhonov analysis of the DEER spectrum as described in the text. Symbols show distance distribution calculated from molecular dynamics using the adaptive biasing force method as described in the text.

144

The distance distribution obtained from DEER spectroscopy may be used to

calculate the mean force between the halves of the coiled coil by analogy with the

method used in potential of mean force (PMF) molecular dynamics (MD)

calculations.175,176 In this type of calculation, the average force Fξ along a selected

“reaction coordinate” ξ (in this case the distance between labeled residues of the coiled-

coil) is related to the derivative of the free energy A(ξ):

( )dA Fd ξξξ

= − [5.2]

Over the course of an MD trajectory, the instantaneous force along ξ is tabulated as a

function of ξ in small bins of width δξ, from which the derivative dA(ξ)/dξ (and thus the

free energy A(ξ)) may be estimated. Optionally, one may apply an adaptive biasing force

(ABF) that cancels the instantaneous force in order to overcome local free energy barriers

and ensure uniform sampling of the ξ coordinate. A(ξ) is then related to the probability

P(ξ) of finding the system at coordinate ξ by the PMF equation:175,176

0)(ln)( APTkA B +−= ξξ [5.3]

Assuming P(ξ) is given by the spin label distance distribution measured by DEER, one

may work backwards from the experimental P(ξ) to find A(ξ) from Equation 5.2, and

then numerically calculate dA(ξ)/dξ to find the average force on the coiled-coil peptide.

Figure 5.13 C compares the experimental measurement of P(ξ) with that

calculated from a 4 ns MD trajectory using the adaptive biasing force (ABF) method as

implemented in the NAMD program [39] (round symbols). The coiled-coil dimer was

145

solvated and equilibrated in a 50 Åx50 Åx70 Å cell using standard methods, and chloride

ions added to neutralize the total charge. The simulations employed the CHARMM27

force-field [40], the particle mesh Ewald method [42] for electrostatic interactions, a

switching function for van der Waals interactions with switching distance of 10 Ǻ and a

cutoff of 12 Å, and the ShakeH [43] algorithm with a to fix hydrogen bond lengths

relative tolerance of 1.0 x 10-8. Calculations were carried out using the Nosé-Hoover NPT

ensemble at 1 atm and 298 K [44] with a damping coefficient of 5 ps and a 2 fs

integration time step. The reaction coordinate ξ was chosen as the distance separating the

centers of mass of the backbone atoms residue 246 in the two chains, and was restricted

to 10 ≤ ξ ≤ 35 Å, allowing the peptide to evolve freely from closed to open state. Since

the DEER experiment measures distances between the electron spins, the curve

calculated from MD curve was shifted by 0.77 nm to take into account the additional

distance between the backbone and the midpoint of the N—O bond of both spin labels

and assist visualization of the two distance distributions.

As Figure 5.13 C shows, the shapes of the calculated and experimental distance

distributions are in excellent agreement. The forces obtained by taking the derivatives of

each curve calculated from the population distributions according to Eq. [5.2] are 110±10

pN for the experimental data and 90±10 pN for the curve calculated from MD. These

values are also quite comparable to typical protein folding and unfolding forces that have

been measured by single-molecule methods.177

It is important to note that the method presented here does require the assumption

that the distribution observed under the conditions of DEER spectroscopy (i.e. in frozen

146

solution) reflects the equilibrium distribution of coiled-coil conformations in solution at

room temperature. This is a reasonably good approximation so long as the freezing is

rapid enough to quench the motions of the system effectively. The availability of model-

independent methods such as Tikhonov analysis for obtaining distributions is another

advantage of the method presented here.

It is also worth emphasizing that accurate force measurements by this method

requires the use of a rigidly attached label such as TOAC; the presence of a flexible tether

as employed in more commonly used spin probes would lead to a broader distance

distribution and systematic underestimation of the forces between the labeled protein

domains. A variety of strategies for immobilizing spin labels relative to the protein have

been employed, including as the attachment of bulky groups on the label that sterically

hinder its motion178 and the use of bifunctional labels that immobilize the nitroxide via

two points of attachment.179

The method described here establishes the utility of EPR for quantitatively measuring

the mechanical properties of peptides and peptide-based nanodevices. Furthermore, these

results add to our understanding of coiled coils motifs, which represent an important and

common mode of protein-protein interaction. EPR has some key advantages for

measuring structural and mechanical properties of coiled-coil motifs: it is uniquely

sensitive to the distance scale that is relevant for the range of motion of such systems,

and, it can measure the full distribution of intramolecular distances, from which

properties including structural flexibility and intermolecular force can be measured. Such

147

capabilities will be critical for the design of nanoscale active devices with targeted

functions.

5.4 SDSL investigation into the structure of UmuD – in collaboration Prof. Penny Beuning, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA

(a) Background UmuD is a manager protein that regulates the activity of Y-family of DNA

polymerases in E. coli. This class of proteins is involved in response to DNA damage and

in regulating DNA replication. UmuD’s function is linked to translesion DNA synthesis

and it relies on a self cleavage activation step and on interaction with other proteins

(Figure 5.14). Understanding the molecular mechanism underlying the action of UmuD is

important for elucidating the cell’s response to mutagenesis. In collaboration with Prof.

Beuning, we are working to elucidate structural features of the UmuD dimer.

We use SDSL to introduce a nitroxide probe at a site of interest as a way to

monitor local dynamics and structure. Relatively little is known about the structure of

UmuD, due to difficulties with crystallization of the uncleaved form. Some studies

suggest that UmuD might be in an intrinsically disordered state 180. Furthermore, it was

suggested that UmuD might form several types of dimers arrangements 181. Our goal is to

further the understanding of the structure of UmuD by adding information about local

dynamics and inter-dimer distance. Understanding UmuD’s structure in solution is

critical to understanding how UmuD regulates the cellular response to DNA damage.

148

Figure 5.14: Schematic representation of UmuD dimer as it undergoes self-cleavage. The star represents the position of the spin labeling site.

(b) Initial results UmuD naturally contains a single cysteine at position 24 that becomes part of a 24-mer

self-cleavage peptide as part of UmuD2 activation. The self-cleavage yields the active

form of, UmuD2`, which interacts with multiple transcription factors. Our first SDSL

experiment is aimed at confirming the proper labeling and establishing that the labeled

protein is still active. In this respect, we used a thiol specific spin label and confirmed

binding by observing lineshape changes. Furthermore, we confirmed retention of self

cleavage by monitoring the appearance of the fast motional signal due to the small

cleavage product. The result of the self cleavage assay are summarized in Figure 5.15.

UmuD2 UmuD2’

Local dynamics Dimer distance

Auto-cleavage

149

Figure 5.15 Stacked EPR spectra of spin labeled UmuD taken at different times where the dotted line highlights the appearance of the fast motional component (i). Plot of the fast motional component as a function of incubation time for pH 7 condition (▲) and for alkaline condition (♦) where the solid lines represent the computed fits (ii).

The appearance of a fast motional component with a pH dependent rate is consistent with

known behavior of UmuD2 self-cleavage activation. This result shows that Cys-24 can be

labeled without affecting the native behavior of the protein. For convenience, it would be

desirable to look at a mutant that does not undergo self-cleavage as a way to investigate

the conformation of the “arms” region of UmuD. The S60A mutant of UmuD is known to

assume the native conformation but does not undergo self-cleavage 181. We spin labeled

the UmuD60A mutant and confirmed that (i) the fast component in not formed, and (ii)

the EPR lineshape is identical to UmuD wild type, suggesting retention of native

structure. The EPR spectra of UmuD-wt and UmuD60A are summarized in Figure 5.16.

346 348 350 352 354 356 358

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 150 200 250 300

Alk data Alk fit pH7 Data pH7 Fit

Time (hours) Magnetic Field (Gauss)

(i) (ii)

150

Figure 5.16: CW-EPR spectra of UmuD-wt (i) and UmuD60A (ii)

Structural information about the dimer conformation of full length UmuD is

limited due in part to flexibility of the cleavable arm region. DEER spectroscopy can be

used to provide information about both structure through measuring interspin distances

and conformation flexibility by measuring distance distribution. Our initial

characterization of spin labeled UmuD60A using DEER spectroscopy has provided some

of the first structural information about this region of the protein. Figure 5.17 summarizes

the results and analysis of DEER spectroscopy. We measure an interspin distance of 40 ±

3 Å and a wide distance distribution consistent with a high degree of conformational

flexibility.

Figure 5.17: DEER spectrum and analysis of singly labeled UmuD 60A.

Magnetic Field (Gauss)

(i)

3440 3460 3480 3500 352 3540 3560 3580 3600

(ii)

Time (µs) Distance (nm) Distance (nm)

3440 3460 3480 350 3520 3540 3560 3580 3600Magnetic Field (Gauss)

151

(c) Ongoing work

The initial results described above provide an encouraging first look at the

applicability of SDSL and EPR to study structural details of UmuD conformation. The

ongoing effort is to prepare mutants with different labeling sites as a way to map the local

dynamics and inter-dimer distances. Additionally a double mutant will also be prepared

to establish the conformation of the dimer and to distinguish between cis or trans

conformers.

152

Chapter 6: Ongoing and Future Work

153

6.1 Distance measurements in ER dimer

(a) Hypothesis of dimer remodeling

In previous chapters, the application of DEER spectroscopy to singly labeled ERα-LBD

was explored. Homodimers spontaneously form both in the unliganded state of ERα-LBD

and in the ligand bound receptor. This makes it possible to measure distances between the

two halves of the singly labeled dimer. In Chapter 3 we described the effects of ligand

binding and coregulator interaction on the dimer distance measured from the 530

position. Similarly, spin labeled estradiol have been used in Chapter 4 to measure

distances between the homodimer. The results have provided for the first time

quantitative measurements of the dimer conformation of ERα-LBD in solution. DEER

measurements, both confirm and elaborate the model put forth from X-ray crystal data.

In particular, these results add the new dimension of distance distribution. We

find that the ERα-LBD dimer is characterized by a substantial degree of conformational

heterogeneity, which is reflected in the wide distance distributions obtained from both

530 labeled ER-LBD and SLE bound receptor. Interestingly, we have observed a marked

change in distance and distance distribution when both agonist ligands and coregulator

peptides are present. This change reduces the inter-molecular distance between the labels

at the 530 position and also considerably reduces the distance distribution suggesting a

more rigid conformation. CW-EPR also confirms this result, showing a marked change in

local dynamics at position 530 upon addition of coregulator peptides. As discussed

154

earlier, these results are consistent with previous fluorescence studies where evidence for

an allosteric stabilization event was presented.

The dimer interface in NRs has been shown to be influenced by ligand and

coactivator binding in the retinoid X receptor LBD (RXR-LBD) heterodimers. In this

case, ligand biding is thought to directly affect the conformation of H11 which constitutes

a large portion of the dimer interface. Similar mechanisms might be regulating the ER-

LBD dimer. Our results allow us to formulate a hypothesis for the effect of ligand and

coregulator peptide binding on the dimer conformation of the receptor.

The unliganded state of ER-LBD is postulated to exist in a number of

interconnecting micro-states, so that as ligand is added, the energetic profile of the

receptor is changed. The energy changes are difficult to characterize experimentally;

however, based on our results we see that ligand interaction does not appreciably affect

the dynamics or the conformation of the dimer as measured by DEER. The ligand does

have an effect on the degree to which peptides stabilize the dimer (c. f. Figure 6.1). This

points to a kinetic effect of ligand binding, where the ligand acts is to reduce the

activation energy (ΔE*) between microstates. In other words, ligand binding does not

affect the relative population between microstates, but it does affect the level of

coactivator recruitment. Even though the apo and holo-ER display similar dynamics and

conformation, they interact differently with the coregulator. The similar dynamics and

conformation points to the fact the relative energy between the microstates is not

significantly affected by ligand binding; however, the observation that the holo state

interacts more efficiently with the coregulators implies that the ligand facilitates

155

conversion to microstates which will be favored by coregulator interaction. These active

conformations are more likely to interact with the coregulator peptide, locking the overall

structure of the receptor. This hypothesis is summarized pictorially in Figure 6.1 where

the case of allosteric interaction in the absence of ligand is also explored.

Figure 6.1: Energy landscape depicting the ligand effect of estradiol binding and coregulator interaction on ER-LBD.

From a structural point of view, it would be desirable to continue the investigation

of the dimer conformation by mapping several distance constraints as a function of ligand

and coregulator interaction. Future work toward this goal should focus on spin labeling of

H11, which constitutes the major dimerization surface in the receptor. Some initial results

have been achieved by looking at inter-dimer distances for ER-543 labeled mutants.

These results are summarized in Figure 6.2. The distances extracted from DEER spectra

of ER-543 dimers agree with crystal structures, but the distance distribution cannot be

accurately derived from these results because of uncertainties in determining the baseline

background. We are currently developing new detection method to address these cases.

E

Degenerate microstates

Apo Holo Holo + CoR

Ligand binding

ΔE* ΔE*

Apo + CoR

coregulator Ligand + coregulator

156

The technical difficulties arise from a combination of factors: (i) fast spin-lattice

relaxation, (ii) broad distributions and (iii) low spin concentrations. The fast relaxation

limits the evolution time available, therefore the slow modulations arising from samples

with broad distance distribution cannot be fully resolved. This leads to difficulties in

calculating the baseline component and increases uncertainty in the measurements. An

average distance can still be measured reliably even for these samples because this

information can be derived from the first part of the spectrum which is less influenced by

incomplete baseline subtraction.

(b) Saturation recovery EPR

Saturation recovery EPR (ST-EPR) is a pulsed EPR technique used to measure dynamics

on the μs timescale. Briefly, in ST-EPR a strong microwave pulse is applied to a portion

of the spectrum creating a saturated “hole”. Since only a portion of the spectrum is being

targeted, only selected orientations will be affected by the pulse. It is then possible to

measure dynamics as these spins rotate causing the hole to spread out at a characteristic

rate. This method could in principle provide important information about the rate of

inter-conversion between different microstates of the receptor.

157

6.2 Double labeled ER

(a) Experimental considerations

In Chapter 2 we described the production and characterization of five ER-LBD mutants,

one of which is designed to be labeled and two sites: position 530 and position 543. This

mutant was created to allow the measurement of ligand dependent changes in

conformation of H12 with respect to the hinge region. DEER spectroscopy is in principle

capable of resolving multiple distances, provided that they are well defined and that a

long enough T2 relaxation time is available to allow sufficient evolution time.

In the case of the double labeled ER-530-543, there are some experimental challenges

which must be addressed. The doubly labeled dimer would in principle provide up to six

different spin-spin distances: two deriving from the intra-molecular interaction of interest

between spins at position 530 and 543, and the remaining four due to inter-molecular

interaction between the dimers. Assuming the halves of the homodimer are equivalent,

this should lead to a maximum of three measurable distance distributions. We have

designed two experiments to help up tease apart the inter- and intra- molecular

contributions: (i) spin dilution, (ii) immobilization to a substrate.

(b) Spin dilution

A simple method to selectively reinforce the relative contribution of the intra-molecular

coupling is by diluting the doubly labeled ER-LBD with unlabeled ER-LBD. This

method relies on equilibrium exchange between ER-530-543 and unlabeled ER-LBD,

and therefore assumes that dimerization is equally probable for homodimers and

158

heterodimers. Figure 6.2 summarizes the spin dilution approach and shows the computed

relationship between the total signal and the intra-molecular signal of interest.

Figure 6.2: Spin dilution scheme highlighting the different combinations of inter and intra-molecular interaction possible within a mixture of doubly labeled and unlabeled ER-LBD (i). Graphical representation showing the dependence of signal intensity (purple), intra-molecular component (green) and relative of intra-molecular component (blue) on the percent dilution (ii).

The graph shows that in order to achieve a 90% contribution of intra-molecular signal, a

60% relative dilution of the doubly labeled ER is necessary, making the ratio of labeled

to unlabeled protein about 1:2.

(c) Immobilization to substrate

As an alternative to spin dilution, ER-LBD decorated beads could be used to artificially

enrich the heterodimer fraction. The method consists of immobilizing unlabeled ER-LBD

to a functionalized bead substrate. This can be accomplished routinely using streptavidin

conjugated beads and biotinylated ER-LBD. Biotinylation is a common protein

derivatization procedure that ensures a strong interaction between the beads and the

Intra-molecular

Intra-molecular

Inter-molecular

Unlabeled – EPR

Spin dilution experiment: signal dependence

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Relative dilution of spin labeled ER (%)R

elat

ive

sign

al (%

)

Total Intra-molecular signalDEER signalWeighted intra-molecular signal

Unlabeled

Double labeled

(i) (ii)

159

modified protein. In fact, the biotin-streptavidin interaction is one of the strongest non-

covalent interactions in nature with a Kd ≈ 10-15 M. This strong interaction will ensure

that ER remains bound to the beads, thus reducing homodimer formation. At this point

the doubly labeled ER-LBD can be introduced, and the excess can be washed away. This

method will yield heterodimer-decorated beads that can be directly used for DEER

experiments. The method is summarized in Figure 6.3.

Figure 6.3: Schematic representation of the production of ER-LBD decorated beads to be used to enrich the heterodimer component of the DEER spectrum.

(d) Initial results

Streptavidin-bead conjugate

Biotinilated ERα-LBD

+

+

Bead decorated with labeled ER heterodimers

160

We cloned, expressed, purified and spin labeled ER-543,530 as previously described.

Figure 6.4 shows the CW-EPR spectra resulting from the doubly labeled mutant. Initial

analysis of the spectrum suggest that it can be easily deconvoluted as a sum of both ER-

530 and ER-543 spectra, suggesting that both sites have been successfully spin labeled

and retain their original conformations. This interpretation is summarized in Figure 6.4.

Currently, we are continuing work with our collaborators at the National High

Magnetic Field Laboratory (NHMFL) in Tallahassee, Florida, to develop a spin dilution

protocol.

Figure 6.4: EPR spectrum of double labeled ER-543,530 (i). EPR spectra of ER-543 (red), ER-530 (green) and result of the addition of the two spectra (dashed) (ii).

3440 3460 3480 3500 3520 3540 3560 3580 3600Magnetic Field (Gauss)

161

6.3 Electrostatic actuation of leucine zipper peptide dimer

(a) Initial results and interpretations

The recent explosion of research in nanotechnology has spurred interest in biologically

inspired nanorobotics,182-184 requiring the development of actuators, joints, motors, and

other machine components on the molecular scale. Because the laws governing their

action may differ substantially from those that govern macroscopic devices, it is

imperative to develop methods to characterize their mechanical properties quantitatively.

In this section we present a preliminary experimental characterization of the action of a

previously proposed design architecture for a novel peptide-based nano-actuator.185-187

The design is based on the α-helical coiled coil leucine zipper portion of the yeast

transcriptional activator GCN4 164 (residues 249-281 of PDB entry 1YSA; hereinafter

GCN4-LZ) which is engineered to obtain an environmentally-responsive device. The

dimeric coiled-coil peptide consists of two identical polypeptide chains ~4.5nm long and

~3nm wide. The actuation mechanism depends on modification of the electrostatic

charges on histidine residues introduced into the peptide, which is achieved by varying

the pH of the solution. Molecular dynamics (MD) calculations have identified specific

amino acid sequences that are predicted to adopt the coiled-coil configuration and to

exhibit reversible opening as a function of ambient pH.185-187

To test the theoretical predictions, peptides were synthesized with a spin label

attached near the N-terminus end open end of each helix, with the objective of forming

the coiled-coil dimer and measuring the distance between the labeled ends by

162

determining the electron spin-spin interaction using electron spin resonance (ESR).166

The TOAC spin label167-169 was selected for this application because of its rigid fused

ring structure, which eliminates motion of the labels relative to the helices, and ensures

that measurements directly reflect the inter-helix distance.

Solid phase peptide synthesis using Fmoc-protection chemistry was performed by

the group of Prof. Gary Lorigan of Miami University (Oxford, OH) on a 433A Peptide

Synthesizer from Applied Biosystems Inc., Foster City, CA. The sequences investigated

are shown in Figure 6.4 together with the structure of the TOAC label. These sequences

were selected based on their Multicoil score165, which indicates the propensity of a

sequence to form a coiled-coil based on the identities of the amino acids in positions a-g of

the coil (cf. Figure 6.4). The control sequence, WT, was constructed as GCN4-LZ with an

N-terminal triglycine added to maintain the same number of residues in all sequences

studied, and to reduce the mobility of the TOAC label167,168,170,171. Sequence M1 is

GCN4-LZ with three histidines at the N-terminus, and M3CT consists of five mutations

of the WT sequence: L253H, K256H, E259H, L261H and Y265H, distributing five

histidines over about half the length of the coil.1 N-terminal trialanine was added to

sequence M3CT to reduce the label mobility. Mutants WT, M1, and M3CT gave average

Multicoil scores of 0.83, 0.92, and 0.93 respectively,188 where a score between 0.5 and 1.0

indicates probable coiled-coil formation. The α-helical content of all samples over the pH

range studied was verified by CD spectroscopy.

163

Figure 6.4: Coiled-coil leucine zipper sequences investigated in this work. Locations of histidine residues substituted into the M1 and M3CT sequences are shown in boldface. Upper right inset: structure of the TOAC spin label. At right is a cartoon of the coiled-coil dimer in its closed conformation, with the TOAC labels explicitly shown.

Figure 6.5 shows CW-EPR spectra from each of the labeled constructs at pH 7.0; the

corresponding spectra at pH 4.0 were effectively identical to these. Whereas the M1

spectrum quite closely resembles that of WT, the M3CT spectrum is considerably

narrower. Dynamic parameters (cf. Fig. 6.5) were obtained by least-squares fitting of the

slow-motional lineshape151,172 with reported magnetic parameters for TOAC.170,173 The

WT and M1 sequences exhibited quite similar anisotropic motion, with the principal

diffusion axis (i.e. the long axis of the dimer) perpendicular to the magnetic x direction

and approximately 30° away from magnetic z, consistent with the direction estimated

from a molecular model. In contrast, the M3CT sample showed much faster and

essentially isotropic motion. These results indicate that M1 most likely forms the same

a

a'

b

b'c

c'

d

d'

e

e'

f f'

g

g'

abcdefg abcdefg abcdefg abcdefg WT GGGXRMKQLEDK VEELLSK NYHLENE VARLKKLVGER M1 HHHXRMKQLEDK VEELLSK NYHLENE VARLKKLVGER M3CT AAAXRMKQHEDH VEHLHSK NHHLENE VARLKKLVGER

ONH

ON

TOAC

164

dimeric structure as the native sequence, whereas M3CT does not form a standard leucine

zipper.

3460 3480 3500 3520 3540 3560 3580

C

B

A

Magnetic Field (Gauss)

Figure 6.5: X-Band CW-EPR spectra of labeled dimers with sequences (A) WT, (B) M1 and (B) M3CT at pH 7.0 and room temperature. Dashed lines show least-squares slow-motional lineshape. Least-squares values of the isotropic rotational diffusion constant R , the rotational anisotropy N, and the βD diffusion tilt were (A) R = 8.7×107 s−1 , N = 95, βD= 25°; (B) R = 4.5×107 s−1 , N = 30, βD= 26°; and (C) R = 1.2×107 s−1, N = 1. Fixed-value parameters in the fits include electronic g-factor (gx, gy, gz) = (2.0085, 2.0056, 2.0020), 14N hyperfine tensor (Ax, Ay , Az) = (21., 9.0, 105) MHz, inhomogeneous Gaussian linewidth 1.9 Gauss, and diffusion tilt angle αD = 90°.

165

Four-pulse double electron-electron resonance (DEER) experiments were performed for

all sequences at pH 4.0 and 7.0 at 65 K. The time domain signals were Fourier-

transformed to give the spectra shown in Figure 6.5 A for each of the sequences studied.

The spectra reflect a frequency distribution given by

( )

3

22210

41cos3

hrgg

πθβμ

ν−

= [6.1]

where g1 and g2 are the isotropic g-factors of each electron, β is the Bohr magneton, μ0 is

vacuum permeability, h is Planck’s constant, r is the interspin distance, and θ is the angle

between the magnetic field and the interspin vector. The spectra exhibit the characteristic

“Pake pattern” with turning points corresponding to θ = 0° and θ = 90° (cf. Eq [6.1]) In

addition, this pattern reflects the distribution of interspin distances r. Figures 6.5 B and

6.5 D shows the distance distribution functions P(r) obtained from the

DEERAnalysis2004 program 189 using a one or two Gaussian distribution model.

The DEER spectrum of WT at pH 7.15 is well described by a single Gaussian

distribution centered at 22 Å, very close to the distance of 23 Å estimated from a

molecular model of the TOAC labeled “closed” zipper conformation (Fig. 6.6). At pH 4.0

a second population appears that can be fit using a Gaussian distribution centered at 36 Å,

corresponding to an open conformation. At pH 7.15, M1 also appears predominantly in

the “closed” configuration (22 Å) with a small population at a longer distance evident in

the Pake pattern At pH = 4.0, most of the population shifts to a second distribution

centered at 43 Å. The DEER spectra of M3CT indicates two components with more open

166

structures than WT or M1 at both pH values, consistent with the result from cw-ESR that

this sequence does not adopt the same coiled-coil structure as the other sequences.

DEER spectra were also analyzed using the model-independent Tikhonov

regularization190. This procedure gave a narrow distribution at 22Å for each of the closed

configuration samples. For M1 at low pH, it gave a broad and somewhat irregular

distribution, consistent with the presence of an open configuration, but difficult to

quantify in terms of open vs. closed populations. To validate our interpretation of the

two-Gaussian model, the relative intensities of the open and closed distributions were

titrated vs. pH, as shown in Fig. 6.6 E. The results are well approximated by a simple

two-state equilibrium model (solid line), suggesting that the two-Gaussian distribution

model is adequate to characterize the state of the device.

Our results clearly demonstrate the possibility of constructing peptide-based

environmentally responsive nanoactuators, and establish the utility of EPR for assessing

the activity of such devices and quantitatively measuring their mechanical properties.

EPR has several key advantages for this application. First, it is uniquely sensitive to the

distance scale that is relevant for the range of motion of such devices. Secondly, it can

measure the full distribution of intramolecular distances, from which such properties as

structural flexibility and actuating force can be measured. Such capabilities will be

critical for the design of nanoscale active devices with targeted functions.

167

pH = 4.0

pH = 7.0

A

pH = 4.0

pH = 7.0

B

-20 0 20

pH = 4.0

pH = 7.0

C

Frequency (MHz)0 2 4 6

pH = 4.0

pH = 7.0

Distance (nm)

D

4 5 6 7 8

0.5

0.6

0.7

0.8

0.9

f clos

ed

pH

Figure 6.6:: DEER results. Rows 1 and 2, WT pH 7 and pH 4. Rows 3 and 4, M1 pH7 and pH 4. Rows 5 and 6 M3pH7 andpH4

168

(b) Difficulties with initial interpretation

Our initial interpretation used normalized DEER spectra as the basis for deriving distance

distribution for the coupled spins. This approximation was justified by the fact that the

sample concentration was kept constant within each pH titration experiment.

Furthermore, the peptide sequence in known to form exclusively coiled-coil dimers in the

pH region tested. This approximation does not affect the validity of the distances

measured at pH 7.0; however, since we are interested in quantifying two different

components (open vs closed conformations), it is important to verify the assumption in

the raw, non-normalized DEER spectra.

We encountered some difficulties reconciling DEER signal intensities when

comparing samples with same spin concentration but different pHs. It appears that as the

pH was titrated from 7 to 4 the DEER signal would increase in intensity. Since the DEER

signal depends on the fraction of coupled spins, the most obvious explanation is that the

amount of spin coupling is increasing at low pH. In other words, if at pH 7 each spin is

interacting with the complementary spin in the coiled-coil dimer, we observe that at

lower pH each spin is interacting with more than one conjugate spin. This is usually the

case with systems undergoing polymerization or aggregation, where more than one

interaction is possible. This hypothesis seems to be supported by a simple test shown in

Figure 6.6, which shows the ratio of the signals from M1 at pH 4 and at pH 7. The result

(curve 3) shows that the modulation due to the short distance is completely removed and

the second component is left unaltered. This implies that the fraction of closed spins does

169

not change, and the additional component is due to a second, pH dependent, population

of interacting spins. Physically this could be explained with peptide tetramers.

Aggregation or polymerization is difficult to rationalize, particularly if we

consider that the peptide become positively charged at low pH and should tend to repel

itself. An alternative explanation is the effect of orientational distribution of the nitroxide

axes with respect to each other. Molecular modeling and our cw-EPR dynamic studies

confirm that the coiled-coil conformation is retained for in the labeled peptide. The

symmetry of the coiled-coil defines precise orientations of the two coupled nitroxides

with respect to each other. The N-O bond axes (e.g. the magnetic x axis) are pointed 180 o

away from each other because of the C2 symmetry of the coiled-coil, and the z axes are

tilted ~30o away from the coiled-coil long axis. As described in Chapter 2, the DEER

signal is acquired by selecting different orientation for pump (x,y) and observe (z)

frequencies. The intensity of the signal depends on the transfer of magnetization between

the two orientations in a system of coupled spins which depends on the orientations of the

two spins with respect to each other. We can think of the spectra as being composed of

two populations of spins: the close one is highly ordered with small spin-coupling

efficiency due to the fixed orientation of the nitroxides, and the open one with a less

ordered interspin orientation and higher coupling efficiency. In this scenario the open

conformation will contribute relatively more to the spectrum intensity.

Currently it is unclear which model better represents the reality of the system.

Other experimental parameters such as cryo-protectants, ionic strength, amino acid

sequence and label position should be explored. Nonetheless, the model and the idea of

170

creating biologically inspired nano-devices that can be controlled is an exciting one and

worth pursuing.

Figure 6.6: Analysis of the raw DEER signal for M1: M1 at pH 7.0 (1), M1 at pH 4.0 (2), result of quotient between curve (2) and curve (1) (from a private communication with Peter Borbat, ACERT, Cornell University, Ithaca, NY).

171

6.4 Initial characterization of 11β spin labeled estradiol

In Chapter 4 we describe the characterization of two spin labeled estradiols using EPR

and fluorescence spectroscopy. The results obtained from that study clearly demonstrate

the utility of an estradiol-nitroxide conjugate that can specifically bind ER and report

details about local environment, structure and binding interaction. The two compounds

described, HO-2105 and HO-2447, can be synthesized using well established techniques

but it is possible to construct SLE with higher affinity by placing the nitroxide derivative

at the 11β position of the estradiol scaffold. Figure 6.7 shows the structure of AH-5-20, a

compound produced by J. Adam Hendricks in Prof. R. Hanson’s laboratory at

Northeastern University, Boston. This compound is expected to bind ER with higher

affinity compared to the 17α or 16 substituted compounds previously described.

O NH O

OH

OHNNHN

N

Figure 6.2: Structure of AH-5-20

An initial characterization of the binding of AH-5-20 to ER-LBD was carried out.

Figure 6.x shows a stacked plot of AH-5-20 in solution (top) and AH-5-20 in the

presence of ER-LBD. The changes observed are characteristic of a decrease in rotation

172

and an increase in the anisotropy of the rotation experienced by the nitroxide probe. This

is consistent with the SLE binding to ER-LBD.

Figure 6.3: cw-EPR spectra of AH-5-25 unbound (top) and bound to ER-LBD (bottom)

3440 3460 3480 3500 3520 3540 3560 3580 3600Magnetic Field (Gauss)

173

Figure 6.9: EPR spectra and lineshape fits (dotted line) for AH-5-20 unbound (i) and bound to ER-LBD (ii). The table summarizes the rotational and magnetic parameters calculated for the two cases. Table 6.1: Lineshape fitting parameters, where the rotation parameters (Rx,y,z) are reported as negative log of the correlation time and the hyperfine values are reported in Gauss. Rotational parameters [-log(τc)] Common magnetic parameter Unbound (i) Bound (ii) g-tensor A-tensor Rx Ry Rz Rx Ry Rz gx gy gz Ax Ay Az 8.9 9.1 8.1 8.68 6.6 7.99 2.009 2.006 2.002 5.3 5.9 37.8

We used NLS lineshape fitting to extract rotational parameters from the EPR

spectra. The results are summarized in Table 1 and are consistent with binding of the SLE

to ER-LBD. In particular, we notice an increase in the anisotropy of the rotation upon

binding that places the fast axis of rotation in the magnetic x direction of the nitroxide.

From these initial results we can conclude that the compound AH-5-20 binding to ER-

LBD and in the bound state the major axis of rotational diffusion is close to N-O bond

axis. Figure 6.11 shows the relevant interaction of estradiol within the binding pocket 37.

The compound AH-5-20 is not expected to disrupt these important interactions.

3440 3460 3480 3500 3520 3540 3560 3580 3600-5

-4

-3

-2

-1

0

1

2

3

4

5 x 10 4

3440 3460 3480 3500 3520 3540 3560 3580 3600-3

-2

-1

0

1

2

3x 104

(i) (ii)

174

Figure 6.10: Coordination of estradiol within the binding cavity; the fast rotation axis of the nitroxide is depicted along the N-O bond in accordance with NLS fitting results.

6.5 Challenges

All the experiments described in this thesis require optimization of a number of

parameters. The next sections summarize some of the most crucial and challenging

obstacles faced.

(a) ER stability

Estrogen Receptor is normally found associated with HSP90 when unactivated by ligand

binding. This suggests that the receptor protein might be prone to unfolding or

aggregation. During the course of this study one of the major hurdles has been to

combine high protein yield with production of active protein. It was noted early on, that a

large amount of expressed protein would be lost to inclusion bodies. In order to minimize

these losses, several induction and extraction conditions were explored. IPTG

H2O

O N H O

O H

OHN

N H N

N

Glu 535 Arg 394

His 524

175

concentration and induction time were adjusted to reduce the level of overexpression.

This helped to increase the concentration of soluble protein fraction at extraction.

The extraction condition was also optimized. We found that the commercially

available extraction buffer, CellLyticX (Sigma), would have to be diluted at least 10X in

order to prevent solubilization of inactive protein.

Purification was greatly aided by selection of a His-tagged plasmid construct. We

found that cold room conditions were necessary to prevent losses due to aggregation.

Considerable precipitation was usually found after the first purification step with Ni-NTA

resin. Closer examination of the pellet by SDS-PAGE shows enrichment of contaminants

compared to the soluble fraction. This prompted the addition of a second cation exchange

purification step that removed the remaining impurities. This procedure also seemed to

improve stability and receptor activity as measured by [3H]estradiol binding.

(b) ER activity assay

The most common assay of estradiol binding is [3H]estradiol binding. In the course of

this study we produced at least one ER-LBD mutant (ER-543) that was not previously

described and therefore needed to be characterized to establish retention of binding

affinity. The assay consists in measuring the bound [3H]estradiol as a function of protein

concentration. The bound radio labeled estradiol is separated from the unbound by adding

a slurry of Dextran coated charcoal to the reaction mixture. The charcoal binds the free

estradiol and is usually separated from the bound estradiol by centrifugation or filtration.

Our attempts to establish the same protocol in-house were unsuccessful. One of the

obstacles encountered was the lack of a dedicated centrifuge for radioactive samples.

176

This limited our options to using filtration in order to separate the charcoal from the

protein mixture. We found that the protein would not filter efficiently through the

membrane and therefore it was impossible to estimate a binding curve. Future attempts

should include use a dedicated centrifuge that has the advantage of decreasing the cost of

analysis and the technical difficulty of the procedure.

6.6 Broader Impact

The results provided here demonstrate how measuring protein flexibility by

SDSL-EPR can significantly enrich the static picture of ligand-receptor interaction

available from crystal structures and more generally add details to the response of protein

conformation to external stimuli. During the course of this thesis we have examined local

changes in dynamics and structure of ER in response to ligand and allosteric binding. We

have also looked at new ways to probe structural flexibility using SLE that offer for the

first time a tool for establishing the dynamics of ligands within a receptor’s binding

pocket. These investigations provide the first quantitative solution structure

measurements for ER-LBD. Although the mean distance constraints derived from DEER

results match well with crystal structures, our measurements add an important new level

to the emerging picture of ligand induced structural remodeling that was previously

unavailable. Based on these results we were able to formulate a new theory of receptor-

ligand interaction for NRs that involved successive levels of structural remodeling that

directly regulate biological action. The broader impact of this work goes beyond

understanding a general model of ligand-receptor interactions; in fact, the results suggest

new pharmacological avenues that can be used to target aberrant NR behavior. The strong

177

effect of allosteric interaction described in Chapter 3 points to this step as being

necessary to direct the biological response of NR. In this respect, it would be desirable to

design compounds that specifically disrupt this interaction such as the peptide-mimetic

compounds being developed in Prof. Hanson’s laboratory. The potential advantage of

such an approach is in being able to target specific pathways of ER action while allowing

the normal functioning to continue undisturbed. Our direct evaluation of allosteric

binding using SDSL-EPR would offer a unique tool to establish the structural effect of

coregulator binding to ER.

The central topic of structural flexibility is an integral part of how receptors and

ligands interact. In Chapter 4 we described some very unique spin labeled estrogens that

introduce a nitroxide directly into the ER’s binding pocket. In addition to being useful

reporters of ligand interaction, we used these probes as structural tool by looking at the

distance distribution occurring between the ER dimers. The initial results reported here

offer an interesting glimpse into some future applications of spin labeled estradiols.

These compounds can be used as ER selective relaxing agents for MRI applications

where estrogen responsive breast cancer cells need to be visualized. SLE also will prove

important for future investigation of ER structure, in fact, the emerging technique of spin

enhanced NMR can be used to elucidate structural folds of the ligand-receptor complex.

Our investigation also looked at effects of environmental factors on structural

flexibility. We looked at hydrophobic mismatch using CB1-TM7 as a model and

electrostatic effects using leucine zipper peptide dimer. In both case we found order-

disorder transitions than can be directly quantified by both CW-EPR and DEER

178

spectroscopy. Taken together, the approaches described in this work are significant in the

way that elements from traditionally separate disciplines such as medicinal chemistry,

magnetic resonance and physical chemistry have been combined to answer specific

questions that are not easily accessible by conventional methods. In this light, the major

impact of this work is the coming together of several philosophies of scientific inquiry,

with the common goal of measuring experimentally protein flexibility.

179

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