Applications of Synchrotron Radiation in Biology and Biotechnology Zehra Sayers Sabanci University, Turkey Chair, SESAME Scientific Committee UPHUK III Bodrum, Turkey Sept. 17 -19, 2007
SYNCHROTRON RADIATION (SR) Acceleration of charged particles results in emission of electromagnetic radiation. H. Winick Initially thought as nuisance because of energy loss from accelerated particles. Importance recognized by early ’ 60 s.
SR: Production 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: Basic properties High flux and brightness Pulsed 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 , mm 2 (a measure of concentration of the radiation)
SR: Storage rings, bending magnets and insertion devices Continuous spectrum characterized by ec = critical energy bending magnet - a “sweeping searchlight” ec(ke. V) = 0. 665 B(T)E 2(Ge. V) eg: for B = 2 T E = 3 Ge. V ec = 12 ke. V wiggler - incoherent superposition (bending magnet fields are usually lower ~ 1 – 1. 5 T) Quasi-monochromatic spectrum with peaks at lower energy than a wiggler 1 = u 2 g 2 (1 + U K 2 )~ (fundamental) 2 g 2 + harmonics at higher energy undulator - coherent interference 0. 95 E 2 (Ge. V) 2 u (cm) (1 + K ) 2 K = g where is the angle in each pole e 1 (ke. V) =
SR: Practical Production and Delivery to Users the storage ring circulates electrons and where they are bent - synchrotron radiation is produced klystrons generate high power radiowaves to sustain electron acceleration, replenishing energy lost to synchrotron radiation electron gun produces electrons (at e. g. 80 ke. V) beam lines transport radiation into “hutches” where instrumentation is available for experiments special “wiggler” insertion devices used to generate x-rays linear accelerator/booster accelerate e- which are transported to storage ring (at e. g. 7 Ge. V)
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. FUNCTIONAL ORGANIZATION • Examples: Chromatin fibre dynamics Cytoskeletal dynamics
SCHEME FOR FUNCTIONAL STUDIES Structural Biology Experimental Methods Modelling Bioinformaics Conservation analysis Cluster analysis Molecular biology Site directed mutagenesis Activity measurements Enzyme kinetics Ligand interactins Activity under perturbation Test structural models Make functional predictions Test functional predictions Make 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 using Microcrystals Large unit cell crystals Cryo-crystallography Minimizing radiation damage Improvement of data quality Automated crystal mounting robot Crogenic robotic crystal transfer system Fed. Ex 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. Mac. Kinnon, “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 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. 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
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). Imaging and Spectroscopy 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
X-RAY SCATTERING AND DIFFRACTION FROM NONCRYSTALLINE SYSTEMS Low 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 imaging Phase contrast imaging Fluorescence Imaging Full field imaging Diffraction enhanced imaging Topography Tomography 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) = ( 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 r 2 p(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 nm r 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 k. Da 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.
durum WHEAT METALLOTHIONEIN Amio acid sequence: MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEV AAGQSGEGCSCGDNCKCNPCNC AAGQSGE Hinge N-terminal C-terminal region Domain b-domain 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. M etal-binding domains are connected by a 42 residue hinge region. Prepare recombinant proteins GSTd. MT and d. MT. Balcali wheat can tolerate higher levels of Cd in soil than C-1252. Bacteria expressing recombinant d. MT can tolerate high levels Cd in growth medium.
MODELING THE STRUCTURE of d. MT Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (b- and a-domains). The predicted 3 D structure of d. MT. 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. Bilecen et al. , 2005
PREPARATION AND CHARACTERIZATION of GSTd. MT Size-exclusion chromatography GSTd. MT elutes as dimer UV Absorbance Measurements Charge transfer band between 250 and 260 nm due to Cd-thiol interactions SDS-PAGE Analysis Native-PAGE Analysis Dynamic Light Scattering (DLS) Measurements
PREPARATION AND CHARACTERIZATION of d. MT Size-exclusion chromatography SDS-PAGE Analysis Native-PAGE Analysis UV Absorbance Measurements Dynamic Light Scattering (DLS) Measurements
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 I 0 is monitored. • IT is the intensity of the beam transmitted through the sample and IT = I 0 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 = 2 Sin /λ where 2 is the scattering angle and λ is the wavelength
BASIC SAXS DATA REDUCTION X 33 camera of EMBL Hamburg Outstation on DORIS STORAGE ring of DESY, Hamburg. Data are collected and reduced using standard software Reference measurements are made on solutions of bovine serum albumin.
Structural models can be calculated ab initio using software such as GASBOR, SASHA etc and rigid body modelling using MASSA, ASSA etc (EMBL-Hamburg)
METALLOTHIONEINS 6 -8 k. Da 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.
durum WHEAT METALLOTHIONEIN Amio acid sequence: MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEV AAGQSGEGCSCGDNCKCNPCNC AAGQSGE Hinge N-terminal C-terminal region Domain b-domain 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. M etal-binding domains are connected by a 42 residue hinge region. Prepare recombinant proteins GSTd. MT and d. MT. Balcali wheat can tolerate higher levels of Cd in soil than C-1252. Bacteria expressing recombinant d. MT can tolerate high levels Cd in growth medium.
MODELING THE STRUCTURE of d. MT Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (b- and a-domains). The predicted 3 D structure of d. MT. 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. Bilecen et al. , 2005
PREPARATION AND CHARACTERIZATION of GSTd. MT Size-exclusion chromatography GSTd. MT elutes as dimer UV Absorbance Measurements Charge transfer band between 250 and 260 nm due to Cd-thiol interactions SDS-PAGE Analysis Native-PAGE Analysis Dynamic Light Scattering (DLS) Measurements
PREPARATION AND CHARACTERIZATION of d. MT Size-exclusion chromatography SDS-PAGE Analysis Native-PAGE Analysis UV Absorbance Measurements Dynamic Light Scattering (DLS) Measurements
SAXS DATA from GSTd. MT 1. Data collected from a 1. 5 mg/ml GSTd. MT solution at X 33 camera on DORIS storage ring. EMBL Hamburg Outstation. GSTd. MT exists as a dimer in solution. The monomer has an extended structure.
ab initio SHAPE DETERMINATION of GSTd. MT Low-resolution GSTd. MT structural model (GASBOR) GST molecules are located in the center of the dimer and d. MT molecules extend from the center.
SAXS DATA from d. MT 1. 1. 0 mg/ml d. MT solution. Experiments are possible only on SR source. d. MT exists as a dimer in solution with an extended structure.
ab initio SHAPE DETERMINATION of d. MT Asymmetry in the structure of d. MT? Implications for Cd-binding? Domain folding? Functional implications.
FUTURE OUTLOOK Macromolecular 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. Spectroscopy Infrared microspectroscopy. EXAFS and imaging. Imaging and spectroscopy. 3 D tomography. Imaging single particles…. .
Useful information can be found at: 1. SSRL website: www-ssrl. slac. stanford. edu 2. www. lightsources. org
ACKNOWLEDEGEMENTS Sabanci University F. Dede G. Dinler F. Kisaayak U. Sezerman H. Budak O. Gokce I. Cakmak SESAME Z. Hussain S. Hasnain G. Vignola H. Winick EMBL Hamburg M. H. J. Koch D. Svergun M. Roessle A. Round M. V. Petoukhov
Скачать презентацию Applications of Synchrotron Radiation in Biology and Biotechnology
7058879a9f95d5618018563c18ba7409.ppt