Determining Protein Structure

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. 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 1H atoms  3-D molecular model Requires: Isotopes (1H, 13C, 15N) d Determining Protein Structure

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

5. Crystals • Well-Ordered • Large Size (~ 0.5 mm) • “Pure” protein at high concentration HOW: Solution Aggregate ↑ Reducing Protein Solubility Amorphous Ordered Crystal

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

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- Low energy X-rays

10. Detectors • 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

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

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

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 Hg2+ Pt+ }

20. Image is formed by applying a mathematical relation • Fourier Transform Spot  Wave of e- density ↲ ↳ Amplitude Phase ⇩ ⇩ Heavy Atom √(Intensity) ⇩ MIR • Difference Patterson Map • 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 • 1-1.5 Å: atoms L I

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 (1H or 15N) possess an intrinsic spin angular momentum, resulting in a magnetic moment m 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 • Relaxation to equilibrium  emits RF that can be measured ↓ High [with respect to a reference] B RF ↓ Nucleus Chemical shifts “Specific” Environment Low For Unique Assignments: Multi-Dimensional NMR: 2-D, 3-D, 4-D

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, I.e. within the same a.a.) • NOESY (NOE, Nuclear Overhausser Effect) • Distance between H atoms close together in space (≤ 5 Å)

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

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

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