Determining Protein Structure BIBC 100

Transcription

1 Determining Protein Structure BIBC 100

2 Determining Protein Structure X-Ray Diffraction Interactions of x-rays with electrons in molecules in a crystal NMR- Nuclear Magnetic Resonance Interactions of magnetic field (external) with the intrinsic magnetic properties of atomic nuclei which possess a spin angular momentum

3 Determining Protein Structure X-RAY Protein Crystal Virtually any size Few, high quality crystals Result: Electron-density map atomic model Requires: Phase determination of the diffracted beams for which heavy metals are used NMR Spectroscopy Limited (2009) ~ 30,000 Da. Protein in High Conc. Result: Distance constraints between 1 H atoms 3-D molecular model Requires: Isotopes ( 1 H, 13 C, 15 N) d

4 Determining Protein Structure X-RAY Frozen Structure NMR Time Resolved Dynamic Folding BOTH OF THEM NEED: Purified Protein Amino Acid Sequence Computers: Molecular Mechanics Molecular Dynamics Molecular Graphics Recombinant DNA Technology

5 Crystals 1. Well-Ordered 2. Large Size (~ 0.5 mm) 3. Pure protein at high concentration HOW: Solution Aggregate Reducing Protein Solubility Amorphous Ordered Crystal

6 Crystals Methods Hanging-Drop: Increase Conc. By vapor diffusion Microdialysis: Increase aggregation by loss of solvent Variables ph Temperature Gravity [protein] solvent

7 Requirements Crystals The repeating unit of a crystal, corresponding ~ to the volume occupied by a single molecule is called a unit cell A crystal is built by billions of identical unit cell X-Rays Electromagnetic radiation of wavelength 1.54 Å They are produced by a beam of accelerating e- on a copper anode target High energy e- X-rays Low energy

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10 Detectors a. Radiation destroys crystals i.e. Protection cooling, rotation, brief exposure, large area detectors b. Records of diffracted (scattered) beams are obtained on - film (x-ray): blackening of emulsion - electronic, solid state, detectors - large area detectors (electronic counters) UCSD c. Comparison of diffraction pattern of native protein crystal with complex of protein and a heavy metal

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12 Braggs Law: 2dsinθ = d = distance θ = reflection angle = wavelength Dictates the conditions for diffraction

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14 Braggs Law: 2dsinθ = d = distance θ = reflection angle = wavelength Dictates the conditions for diffraction

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17 Phase Determination Problem Each diffracted beam is defined by: Amplitude = intensity of spot-measure Wavelength = you know Phase = lost in experiment Microscope out of focus/no eyepiece Solution: Multiple isomorphous replacement Diffusion of heavy metals into channels } SH groups reactivity Replacement of light metals Hg 2+ Pt +

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20 Image is formed by applying a mathematical relation Fourier Transform Spot Wave of e- density Amplitude Phase (Intensity) Heavy Atom Difference Patterson Map MIR To determine position of heavy atom in crystal To determine phase of heavy atom in crystal Use 2 different heavy atoms to decrease ambiguity

21 Calculation of the electron density map Interpretation of the electron density map Resolution: Quality of crystal (~2 Å) 5-6 Å: Course of polypeptide chain ~3 Å: side chains, e.g. ~ 2 Å: side chains Å: atoms L I

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26 Refinement: Model Building R factor: residual disagreement, between a hypothetical crystal containing the model and the experimentally determined crystal R = 0 perfect agreement R = 0.59 total disagreement For a resolution of 3Å and R 0.3? For 2Å, R=0.2 OK

27 NMR Atomic Nuclei ( 1 H or 15 N) possess an intrinsic spin angular momentum, resulting in a magnetic moment that can interact with an externally applied magnetic field B In a B field the spins of H align Equilibrium alignment can be perturbed by pulses of radiofrequency (RF)

28 NMR B Relaxation to equilibrium emits RF that can be measured High Low RF [with respect to a reference] Nucleus Chemical shifts Specific Environment For Unique Assignments: Multi-Dimensional NMR: 2-D, 3-D, 4-D

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30 NMR 2-D NMR Diagonal: ~ 1-D spectrum Peaks off-diagonal : (cross-peaks) Interactions of H atoms that are close to each other in space COSY: (Correlation) Fingerprint of a.a. Distance between BONDED H atoms ( three chemical bonds, namely within the same a.a.) NOESY (NOE, Nuclear Overhausser Effect) Distance between H atoms close together in space ( 5 Å)

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33 Interpretation Sequence Specific Assignment or Sequential assignment 1. COSY Cross peak unique for each amino acid, fingerprint Which a.a. in sequence? 2. NOESY a. interactions in space { Cross-peaks b. interactions of residues that are sequentially adjacent (i and i+1) 3. Amino Acid Sequence 4. Distance Constraints: for H atoms in i to H atoms in j

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35 Interpretation Sequence Specific Assignment or Sequential assignment 1. COSY Cross peak unique for each amino acid, fingerprint Which a.a. in sequence? 2. NOESY a. interactions in space { Cross-peaks b. interactions of residues that are sequentially adjacent (i and i+1) 3. Amino Acid Sequence 4. Distance Constraints: for H atoms in i to H atoms in j

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44 Interpretation cont 5. Structure Refinements - computer modeling - No unique structure but different structures that are compatible with data (ambiguity)

45 By Combination of x-ray and NMR Structure Determination Complementary In general agreement (except minor discrepancies especially loops) Both require: Biochemical Information Supercomputer Processing

46 Determining Protein Structure X-Ray Diffraction Interactions of x-rays with electrons in molecules in a crystal NMR- Nuclear Magnetic Resonance Interactions of magnetic field (external) with the intrinsic magnetic properties of atomic nuclei which possess a spin angular momentum COMPLEMENTARY