Paramagnetic Effects

BCMB/CHEM 81902012

ReferencesExpanding the utility of NMR restraints with paramagnetic compounds: Background and practical aspects, Koehler J and Meiler J,Prog. NMR Spect. 59: 360-389 (2011)Paramagnetic tagging for protein structure and dynamics analysis, Keizers PM and Ubbink M, Prog. NMR Spect. 58: 88-96 (2011) Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation, Clore GM, Prot. Sci. 20: 229-246 (2011) Lanthanide-tagged proteins - an illuminating partnership, Allen KN and Imperiali B, Curr. Opin. Chem. Biol. 14: 247-254 (2010) Protein NMR Using Paramagnetic Ions, Otting G, Ann. Rev. Biophys, 39: 387-405 (2010) Paramagnetic labelling of proteins and oligonucleotides for NMR, Su X-C and Otting G, J. Biomol. NMR, 46: 101-112 (2010)

Paramagnetic Dipolar Spin Relaxation Effects

])(1

61

3)(1

[)1()()4

(152

2222226

22H2

1cSH

c

cH

c

cSH

cBeop r

SSgRτωω

ττω

ττωω

τμγπμ

+++

++

−++

=

])(1

6)(1

61

3)(1

4[)1()()4

(151

2222

22226

22H2

2

cS

c

cSH

c

cH

c

cSH

cc

Beop r

SSgR

τωτ

τωωτ

τωτ

τωωτ

τμγ

πμ

++

+++

++

−++

+=

• τc-1 = τm

-1 + τe-1 where τm and τe are molecular

tumbling and electron spin correlation times• Shortest term dominates, τe can add field dependence• ωS is large at 11.7T (2x1012)•τc of 10-9, ωH, τc terms dominates, R1 < R2

Relaxation Enhancement by Free Radicals (Nitroxides) can Identify Interaction Sites.

Example: Galectin Interacting with LacNAc

NO.

OHONN

N

O

NO.

OO

Dhbt-OH

THF, DCC DMF, DIPEA

O

AcNHHOO

OH

O

OHHO

HO OH

NH2

O

AcNHHOO

OH

O

OHHO

HO OH

NHO N

CH3

CH3CH3

O.CH3

Synthesis of a Spin-Labeled N-acetyllactosamine

15N1H

Intensities in HSQC Experiments are Measures of R2(transverse proton magnetization during 2τ period is major loss)

1H(I)

15N(S)

Battiste and Wagner (2000) Biochemistry 39:5355-5365

90-x 180y 90y

90x

τ ττ = 1/4J

Change in 15N HSQC spectrum (800 MHz)ofGalectin-3 upon addition of LacNac-TEMPO

0 mM 10 mM

Distances from R2 Equation

Residue X-Ray model (Å)a

Spin Label method (Å)b

τc = 6 ns τc = 8 ns τc = 10 ns

182 14.0 14.9 15.6 16.2

184 11.9 10.7 11.2 11.6

185 14.2 14.9 15.5 16.1

186 14.9 13.7 14.4 14.9

187 17.2 17.2 18.0 18.6

162 18.9 17.4 18.2 18.9

164 19.1 19.5 20.4 21.1

X-Ray crystal structure of Galectin-3 (Seetharamana et al. 1998)

E184

R186

E165

A245

K227

There are some anomalies – control looking at TEMPO alone

Bicelle

Proteins Can Also Be Tagged

Membrane-Bound myr-yARF1-GTP

Membrane model: DHPC/DMPC bicelles.

Complex ~ 70kDA

PREs Give Long-Range Distance Constraints

∑∑= =

−− =∝e mN

i

N

jij

me

rNN

rPRE1 1

66 1

+

N O

15 N-1 H

r

PRE Data using 1H-15N HSQC AttenuationsS62C-MTSL

D67

W66

Fitting PREs Required 3-State Averaging for N-terminal Helix

Ensemble Averaging:XPLOR-NIHover 3 nitroxide conformersover 3 protein conformers

Liu Y, Kahn RA and Prestegard JH (2010). Nature Struct Mol Biol. 17:876-81.

Iwahara,Schwieters,and Clore(2004) JACS, 126, 5879-96

Myr-yARF-GTP – FAPP1-PH Interactions

FAPP paramagnetic perturbations from T56C ARF

180

W15

G67

E80  A78

N58

FAPP‐ARF docked models using PREs

180

90

Membrane

Metal Ions Have useful Paramagnetic PropertiesBertini, Luchinat, Parigi, (2001) “Solution NMR of Paramagnetic Molecules”

Short Electron Spin Lifetimes: Contact Shifts, Pseudo-Contact Shifts and Field Alignment

(Bertini, 2001)

Curie Spin Relaxation Still Occurs for Short τe

)1

34()2(

)1()(Br1)

4(

51

222

2242H

2o

62

rH

rr

B

BJoPRE

TkJJg

τωτ

τμγ

πμ

λ+

++

=

• Comes from excess population of β spin state –there is a net average magnetic moment

• Note field squared dependence – lose signals at 10Å at 18T

• Best to use an ion with low J

Identifying a Dimer Interface with (Gd-DTPA)Lee H-W, Wylie G, Bansal S; et al. Prot. Sci. 19:1673-1685 (2010)

Comparison of crosspeakattenuation at high and low protein concentration. Ratio of effects givens protection factor. Red are protected

Pseudo Contact Shifts and Molecular AlignmentBertini, I., et al. (2002). Concepts in Magnetic Resonance 14: 259-286.

]2cossin)1cos3([B

1201

]2cossin)1cos3([12

1

223

22o

2

223

ΦΘΔ−−ΘΔ=

Δ−−Δ=

rhaxBNH

NH

rhax

TkrS

RDC

rPCS

χχγγ

π

ϕθχθχπ

h

• These effects depend on anisotropic susceptibilities• Size of Induced dipoles depends on molecular orientation

Hence, rotational averaging does not reduce dipolar field tozero in PCS case

• Difference in interaction with B0 for different orientations results in field induced alignment and measureable RDCs

Metal binding peptide sequences (EF-hand)

can be added to protein constructs.

Pseudo-contact shifts and RDCs

provide validation of structure and

positioning of ligands

Lanthanide –Tagged Galectin-3Zhuang T, Lee H-S, Imperiali B et al., Prot. Sci. 17:1220-1231 (2008)

PCSs of Galectin-3-LBT

Lu3+ - BlackDy3+ - Green

15N-1H RDCs and PCSs of Galectin-3-LBT(Dy3+)(Agreement with crystal structure 1A3K)

Agreement of RDCs and PCSs suggest a rigid model can be used

PCSobs(Hz) PCSbound(Hz) Back-cal(Hz)

Glc(α) H1 -10±1 -48±5 -45

H2 -116±1 -53±5 -55

H3 -8±1 -42±5 -46

H5 -10±1 -51±5 -53

Gal H1 -10±3 -50±15 -49

H2 -9±3 --45±15 -49

H3 -9±3 45±15 -43

H4 -11±3 -55±15 -51

H5 -11±3 -55±15 -59

PCSs for 2.5mM Lactose with 0.5mM Galectin-3 at 1H frequency 800MHz