applications of synchrotron radiation in biology and biotechnology zehra sayers sabanci university,...

Applications of Synchrotron Radiation in Biology and Biotechnology

Zehra SayersSabanci University, Turkey

Chair, SESAME Scientific Committee

UPHUK IIIBodrum, TurkeySept. 17-19, 2007

SYNCHROTRON RADIATION (SR)

H. Winick

Acceleration of charged particles results in emission of electromagnetic radiation.

Initially thought as nuisance because of energy loss from accelerated particles.Importance recognized by early ’60s.

At low electron velocity (non-relativistic case) radiation is emitted in a non-directional pattern.

When the electron velocity approaches the velocity of light radiation is emitted in the direction of motion and the radiated power goes up dramatically.

SR: Production

High flux and brightnessPulsed time structure Tunability

Polarized (linear, elliptical, circular)

Small source size

Partial coherence

High stability

Flux = # of photons in given /sec, mrad

Brightness = # of photons in given /

sec, mrad , mrad , mm2

(a measure of concentration of the radiation)

SR: Basic properties

Continuous spectrum characterized by c = critical energy

c(keV) = 0.665 B(T)E2(GeV)

eg: for B = 2T E = 3GeV c = 12keV

(bending magnet fields are usually lower ~ 1 – 1.5T)

Quasi-monochromatic spectrum with peaks at lower energy than a wiggler

1 (keV) =

K = where is the angle in each pole

1 = u

(1 + ) ~ (fundamental)K

2

U

+ harmonics at higher energy

0.95 E2 (GeV)K u

(cm) (1 + )2

undulator - coherent interference

wiggler - incoherent superposition

bending magnet - a “sweeping searchlight”

SR:Storage rings, bending magnets and insertion devices

klystrons generate high power radiowaves to sustain electron acceleration, replenishing energy lost to synchrotron radiation

electron gun produces electrons (at e.g. 80 keV)

linear accelerator/booster accelerate e- which are transported to storage ring (at e.g. 7 GeV)

the storage ring circulates electrons and where they are bent - synchrotron radiation is produced

beam lines transport radiation into “hutches” where instrumentation is available for experiments

special “wiggler” insertion devices used to generate x-rays

SR: Practical Production and Delivery to Users

SR: Biological and Biotechnological Applications

“Biologists” are involved in 4 types of experiments at SR sources:

Macromolecular Crystallography.

Spectroscopy.

X-ray Diffraction and Scattering from non-crystalline systems.

Imaging.

WHAT ARE THE ADVANTAGES OF USING SR TECHNIQUES IN BIOLOGY?

MACROMOLECULES OF LIVING SYSTEMS

• Special architecture at molecular structure level;

Nucleic acids (DNA, RNA), Proteins, Lipids, Carbohydrates.

• Examples:

DNA Proteins

• Hierarchical Organizational at larger scale:

Static and dynamic structures.

Cytoskeletal dynamicsChromatin fibre dynamics

FUNCTIONAL ORGANIZATION

• Examples:

SCHEME FOR FUNCTIONAL STUDIES

Structural BiologyExperimental MethodsModelling

BioinformaicsConservation analysisCluster analysis

Molecular biologySite directed mutagenesis

Activity measurements Enzyme kineticsLigand interactinsActivity under perturbation

Test structural modelsMake functional predictions

Test functional predictionsMake structural predictions

STRUCTURE AND FUNCTION RELATIONSHIP

• Experiments: Static and Dynamic

measurements of structural parameters.

• Calculations: Prediction of structure, structural change where and how.

SR offers a wide selection of powerful experimental tools for determination of structural parameters.

Time resolved data for establishment of correlation between structural change and function.

MACROMOLECULAR CRYSTALLOGRAPHY

Determination of structure of macromolecules at atomic resolution.

Applications include:

Therapeutic drug design Enzyme mechanisms Supramolecular structure Molecular recognition Nucleic acids Structural genomics High-throughput crystallography

SR sources; high intensity, small beam size, and collimation.

The MAD (multi-wavelength anomalous ddiffraction) phasing method readily applicable with tunable radiation at SR sources,

MACROMOLECULAR CRYSTALLOGRAPHY

SR offers possibility of usingMicrocrystalsLarge unit cell crystals

Cryo-crystallographyMinimizing radiation damageImprovement of data quality

Automated crystal mounting robot

Crogenic robotic crystal transfer system

FedEx Crystallography!!!

SSRL, SAM

MACROMOLECULAR CRYSTALLOGRAPHY: Highlights

Nobel Prize 2003

Mechanism for the voltage dependent K-ion channel.

Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon, “X-ray structure of a voltage-dependent K+ channel,” Nature 423, 33 (2003).

Nobel Prize 2007

Mechanism for RNA polymerase II.

Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, P. Cramer, D.A. Bushnell, J. Fu, A.L. Gnatt, B. Maier-Davis, N.E. Thompson, R.R. Burgess, A.M. Edwards, P.R. David, and R.D. Kornberg, “Architecture of RNA polymerase II and implications for the transcription mechanism,” Science 288, 640 (2000).

SPECTROSCOPY

X-ray absorption spectroscopy EXAFS; atomic arrangements, bond distance, coordination no., symmtery,

XANES: valence,

Magnetic circular dichroism: spin-orbit magnetic moments

X-ray fluorescence spectroscopy Quantitative analysis of elemental distribution

Far and near Infra-red, VUV spectroscopy

Vibrational spectroscopy

Hard X-ray spectroscopy:Extended x-ray absorption fine structure (EXAFS) spectroscopy, X-ray absorption spectroscopy (XAS), Near-edge x-ray absorption fine structure (NEXAFS) spectroscopy, X-ray absorption near-edge structure (XANES) spectroscopy, X-ray magnetic circular dichroism (XMCD)

Investigations of geometric and electronic structure.

Sensitive to element, oxidation state and symmetry of the molecules.

Tunability of SR is essential.

Investigation of silent Zn in Metalloenzymes

Zn K-edge EXAFS as a function of time.O. Kleinfeld, A. Frenkel, J.M.L. Martin, and I. Sagi, “Active site electronic structure and dynamics during metalloenzyme catalysis,” Nat. Struct. Biol. 10, 98 (2003).

Investigation of elemetal composition of cancerous lung tissue can be compared with that of healthy tissue by X-ray fluorescence mapping measurements. An optical micrograph of lung tissue is shown together with specific maps showing Fe, Cu and Zn distributions in the boxed area of the tissue.SSRL

Imaging and Spectroscopy

X-RAY SCATTERING AND DIFFRACTION FROM NONCRYSTALLINE SYSTEMSLow resolution data on the size and shape of the molecule can be obtained.

Time-resolved data in response to a perturbation on the system.

Protein solutions, fibers. Biomaterials: membranes, lipid micelles.

Measurements can be made at small (SAXS) and/or wide angles (WAXS) depending on the system.

Complementary data to crystallography, electron microscopy and spectroscopic measurements.

Applications include:Protein (DNA)-ligand interactions.Drug delivery.Material characterization.Time-resolved changes instructure.

X-RAY SCATTERING AND DIFFRACTION FROM NONCRYSTALLINE SYSTEMS

Examples:

Bacterial crystals

Rat tail tendon

IMAGING

Absorption contrast imagingPhase contrast imagingFluorescence ImagingFull field imagingDiffraction enhanced imagingTopographyTomography

X-RAY THERAPY

Targeted and dose-controlled therapy.

CLOSER LOOK SMALL ANGLE X-RAY SCATTERING (SAXS) FROM PROTEIN SOLUTIONS

SMALL ANGLE SOLUTION X-RAY SCATTERING

• Small angle X-ray scattering results from inhomogeneities in the electron density in a solution due to macromolecules dispersed in the uniform electron density of the solvent (0).

A solution of macromolecules

Solute: protein, DNA, polymer (p)

Solvent (0)

• Scattering pattern is determined by the excess electron density of the solute, (r)

(r) = (p-0)c(r) + s(r) = av c (r) + s (r) (1)

Where p = the average electron density of the particle.

av = the average electron density of the particle above the level of the solvent (contrast).

c (r) = dimensionless function describing the volume of the solute (with the value 1 inside the particle and 0 elsewhere).

s (r) = fluctuations of the electron density above and below the mean value (independent of the contrast).

•In an ideal solution all particles are identical and randomly positioned and oriented in the solvent.

•Scattering pattern contains information about the spherically averaged structure of the solute described by a distance probability function p(r)

•p(r) is the spherically averaged autocorrelation function of (r) and r2p(r) is the probability of finding a point inside the particle at a distance between r and r+dr from any other point inside the particle

Dmax

• For a globular particle p(r) has two main regions

a. A region of sharp fluctuations due to neighbouring atom pairs (0.1 nmr 0.5 nm) and of damped oscillations due to structural domains

(i.e -helices in proteins)

b. A smooth region corresponding to intramolecular vectors.

• Beyond Dmax p(r) vanishes

• The scattering curve also contains two regions:

a. Small angle region; information on the long range organization (shape) of the particle

b. Large (wide) angle region; internal structure of the particle (deviations from p)

Large distances only contribute at low angles.

Short distances contribute over a large angular range and at high angles their contribution dominates the scattering pattern.

SCATTERING PATTERN AND THE DISTANCE DIFSTRIBUTION FUNCTION p(r)

Scattering intensity and the distance distribution function are related by a Henkel transformation.

APPLICATIONS

• Determination of radius gyration, radius gyration of the cross section, molecular weight.

• Shape determination; at low angle (2-3 nm) the scattering curve is dominated by the shape of the particle.

• Time-resolved measurements for determination of structural changes during interactions or upon a perturbation on the system.

• Modern methods allow domain structure analysis, possibility of modeling loop domains, analysis of non-equilibrium systems (Svergun and Koch 2002, Current Opinion in Structural Biology, 12:654-660).

METALLOTHIONEINS

6-8 kDa proteins that bind metals in a wide range of organisms.

High cysteine (cys) content (up to 30%) in the amino acid sequence and bind metals through the thiol groups of cys residues.

Metal composition depends on the source and previous exposure to metals. Human liver MT contains mainly Zn, that isolated from kidneys contain Cd and Zn or Cu. In higher organisms MTs represent the only protein that is a natural Cd ligand.

Precise physiological functions are not yet identified; MTs are involved in transport and storage of essential metal ions (Cu and Zn) and detoxification (Cd and Hg).

Durum wheat MT is expressed and synthesized at high levels during exposure Cd.

MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEVAAGQSGEGQFEVAAGQSGEGCSCGDNCKCNPCNCN-terminalN-terminal

DomainDomain-domain-domain

C-terminalC-terminalDomainDomain

-domain-domain

HingeHingeregionregion

C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein

“Cystein motifs” (cys-motifs) are involved in metal binding.Metal-binding domains are connected by a 42 residue hinge region.Prepare recombinant proteins GSTdMT and dMT.

durum WHEAT METALLOTHIONEIN

Balcali wheat can tolerate higher levels of Cd in soil than C-1252.Bacteria expressing recombinant dMT can tolerate high levels Cd in growth medium.

Amio acid sequence:

Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (- and -domains).

Bilecen et al., 2005

The predicted 3D structure of dMT. Cadmium (blue spheres)-binding metal centers at each pole of the dumbbell-shaped molecule are depicted in ball and stick representation with the extended hinge region highlighted in ribbon representation.

MODELING THE STRUCTURE of dMT

Size-exclusion chromatography

Charge transfer band between 250 and 260 nm due to Cd-thiol interactions

UV Absorbance MeasurementsDynamic Light Scattering

(DLS)Measurements

GSTdMT elutes as dimer

SDS-PAGE Analysis Native-PAGE Analysis

PREPARATION AND CHARACTERIZATION of GSTdMT

Size-exclusion chromatography

SDS-PAGE Analysis Native-PAGE Analysis

UV Absorbance Measurements Dynamic Light Scattering (DLS)Measurements

PREPARATION AND CHARACTERIZATION of dMT

EXPERIMENTAL SET-UP FOR SAXS MEASUREMENTS

THE PRINCIPLE OF A SMALL ANGLE X-RAY SOLUTION SCATTERING EXPERIMENT

• The optical system selects X-rays with a wavelength of 0.15 nm and a narrow band-width

• The beam is focused on a position sensitive detector with an adequate cross section at the sample position

• The incident beam intensity I0 is monitored.

• IT is the intensity of the beam transmitted through the sample and IT = I0 exp(-µt), where the factor (-µt) represents the absorbance of a solution of thickness t

• I(s) is the scattered intensity which depends on the scattering vector s defined as

s = 2Sin/λ

where 2 is the scattering angle and λ is the wavelength

X33 camera of EMBL Hamburg Outstation on DORIS STORAGE ring of DESY, Hamburg.Data are collected and reduced using standard softwareReference measurements are made on solutions of bovine serum albumin.

BASIC SAXS DATA REDUCTION

Structural models can be calculated ab initio using software such asGASBOR, SASHA etc and rigid body modelling using MASSA, ASSA etc (EMBL-Hamburg)

METALLOTHIONEINS

6-8 kDa proteins that bind metals in a wide range of organisms.

High cysteine (cys) content (up to 30%) in the amino acid sequence and bind metals through the thiol groups of cys residues.

Metal composition depends on the source and previous exposure to metals. Human liver MT contains mainly Zn, that isolated from kidneys contain Cd and Zn or Cu. In higher organisms MTs represent the only protein that is a natural Cd ligand.

Precise physiological functions are not yet identified; MTs are involved in transport and storage of essential metal ions (Cu and Zn) and detoxification (Cd and Hg).

Durum wheat MT is expressed and synthesized at high levels during exposure Cd.

MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEVAAGQSGEGQFEVAAGQSGEGCSCGDNCKCNPCNCN-terminalN-terminal

DomainDomain-domain-domain

C-terminalC-terminalDomainDomain

-domain-domain

HingeHingeregionregion

C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein

“Cystein motifs” (cys-motifs) are involved in metal binding.Metal-binding domains are connected by a 42 residue hinge region.Prepare recombinant proteins GSTdMT and dMT.

durum WHEAT METALLOTHIONEIN

Balcali wheat can tolerate higher levels of Cd in soil than C-1252.Bacteria expressing recombinant dMT can tolerate high levels Cd in growth medium.

Amio acid sequence:

Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (- and -domains).

Bilecen et al., 2005

The predicted 3D structure of dMT. Cadmium (blue spheres)-binding metal centers at each pole of the dumbbell-shaped molecule are depicted in ball and stick representation with the extended hinge region highlighted in ribbon representation.

MODELING THE STRUCTURE of dMT

Size-exclusion chromatography

Charge transfer band between 250 and 260 nm due to Cd-thiol interactions

UV Absorbance MeasurementsDynamic Light Scattering

(DLS)Measurements

GSTdMT elutes as dimer

SDS-PAGE Analysis Native-PAGE Analysis

PREPARATION AND CHARACTERIZATION of GSTdMT

Size-exclusion chromatography

SDS-PAGE Analysis Native-PAGE Analysis

UV Absorbance Measurements Dynamic Light Scattering (DLS)Measurements

PREPARATION AND CHARACTERIZATION of dMT

s (nm-1)

0.5 1.0 1.5 2.0 2.5 3.0

log

(I)

-3

-2

-1

0

1

2

3

GSTdMT

s2 (nm-2)

0.06 0.08 0.10 0.12

ln (

I)

5.1

5.2

5.3

5.4

5.5

5.6

Guinier plot of GSTdMTLinear Fit

s2 (nm-2)

0.06 0.08 0.10 0.12-0.03

-0.02

-0.01

0.00

0.01

0.02

0.03

GSTdMT exists as a dimer in solution.

The monomer has an extended structure.

SAXS DATA from GSTdMT

Data collected from a1.5 mg/ml GSTdMT solution at X33 camera on DORIS storage ring. EMBL Hamburg Outstation.

GST molecules are located in the center of the dimer and dMT molecules extend from the center.

Low-resolution GSTdMT structural model (GASBOR)

ab initio SHAPE DETERMINATION of GSTdMT

s (nm-1)

0.5 1.0 1.5 2.0 2.5 3.0

Log

(I)

-0.5

0.0

0.5

1.0

1.5

2.0

dMT

X Data

ln (

I)

3.2

3.4

3.6

3.8

4.0

4.2

4.4Guinier plot of dMTlinear fit

s2 (nm-2)

0.20 0.25 0.30 0.35 0.40

-0.3

-0.2

-0.1

0.0

0.1

0.2

SAXS DATA from dMT

1.0 mg/ml dMT solution.

Experiments are possible only on SR source.

dMT exists as a dimer in solution with an extended structure.

Asymmetry in the structure of dMT?Implications for Cd-binding?Domain folding?Functional implications.

ab initio SHAPE DETERMINATION of dMT

FUTURE OUTLOOKMacromolecular crystallography

High throughput crystal structure determination.Automated remote screening and data collection.Time-resolved crystallography.Crystallography and SAXS.

X-ray Scattering

Cryo-SAXS.Time-resolved SAXS.High-resolution micro-beam SAXS.Combination with SRCD.

SpectroscopyInfrared microspectroscopy.EXAFS and imaging.

ImagingImaging and spectroscopy.3D tomography.Imaging single particles…..

Useful information can be found at:

1. SSRL website: www-ssrl.slac.stanford.edu

2. www.lightsources.org

Sabanci UniversityF.DedeG. DinlerF. KisaayakU. SezermanH. BudakO.GokceI. Cakmak

EMBL HamburgM.H.J. KochD. SvergunM. RoessleA. RoundM. V. Petoukhov

SESAMEZ. HussainS. HasnainG. VignolaH. Winick

ACKNOWLEDEGEMENTS