Determining Protein StructureBIBC 100Determining Protein Structure• X-Ray Diffraction– Interactions of x-rays with electrons inmolecules in a crystal• NMR- Nuclear Magnetic Resonance– Interactions of magnetic field (external) withthe intrinsic magnetic properties of atomicnuclei which possess a spin angular momentumDetermining Protein Structure• X-RAY– Protein Crystal– “Virtually” any size– Few, high quality crystalsResult: Electron-density map‡ atomic modelRequires: Phase determinationof the diffracted beams forwhich heavy metals areused• NMR Spectroscopy– Limited (2004) ~ 30,000 Da.– Protein in High Conc.Result: Distance constraintsbetween 1H atoms ‡ 3-Dmolecular modelRequires: Isotopes (1H, 13C, 15N)dDetermining Protein StructureX-RAY• Frozen StructureNMR• Time Resolved• “Dynamic” ‡ FoldingBOTH OF THEM NEED:• Purified Protein• Amino Acid Sequence• Computers:Molecular MechanicsMolecular DynamicsMolecular Graphics• Recombinant DNATechnologyCrystals1. Well-Ordered2. Large Size (~ 0.5 mm)3. “Pure” protein at high concentrationHOW:Solution‡ Aggregate ↑ Reducing Protein SolubilityAmorphousOrdered ‡CrystalCrystals• Methods– Hanging-Drop:Increase Conc. Byvapor diffusion– Microdialysis: Increaseaggregation by loss ofsolvent• Variables– pH– Temperature– Gravity– [protein]– solventRequirements• Crystals– The repeating unit of a crystal, corresponding ~ to thevolume occupied by a single molecule is called a unitecell– A crystal is built by billions of identical unit cells• X-Rays– Electromagnetic radiation of wavelength 1.54 Å– They are produced by a beam of accelerating e- on acopper anode targetHigh energy e- Low energyX-raysDetectorsa. Radiation destroys crystalsi.e. Protection ‡ cooling, rotation, brief exposure, largearea detectorsb. Records of diffracted (scattered) beams areobtained on- film (x-ray): blackening of emulstion- electronic, solid state, detectors- large area detectors (electronic counters) – UCSDc. Comparison of diffraction pattern of nativeprotein crystal with complex of protein and aheavy metalBraggs Law:2dsin_ = l d = distance_ = reflection anglel = wavelengthPhase 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 metalsHg2+Pt+}• Image is formed by applying a mathematicalrelation• Fourier Transform Spot ‡ Wave of e- densityAmplitude Phase√(Intensity) Heavy Atom ⇩ ⇩⇩MIRDifference 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• Calculation of the electron density map• Interpretation of the electron density map– Resolution: Quality of crystal (~2A)• 5-6 Å: Course of polypeptide chain• ~3 Å: side chains, e.g.• ~ 2 Å: side chains• 1-1.5 Å: atomsLIRefinement: Model Building• R factor: residual disagreement, between ahypothetical crystal containing the modeland the experimentally determined crystal– R = 0 perfect agreement– R = 0.59 total disagreement• For a resolution of ≤ 3Å andR ≤0.3 ‡ ?• For ≤2Å, R=0.2 ‡ OKNMR• Atomic Nuclei (1H or 15N) possess anintrinsic spin angular momentum, resultingin a magnetic moment _ that can interactwith an externally applied magnetic field B• In a B field the spins of H align• Equilibrium alignment can be perturbed bypulses of radiofrequency (RF)NMR• Relaxation to equilibrium ‡ emits RF thatcan be measured[with respect to a reference]Chemical shifts “Specific”NucleusEnvironment↓↓HighLowB RFFor Unique Assignments:Multi-Dimensional NMR: 2-D, 3-D, 4-DNMR• 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 chemicalbonds, I.e. within the same a.a.)• NOESY (NOE, Nuclear Overhausser Effect)– Distance between H atoms close together in space (≤ 5Å)Interpretation• Sequence Specific Assignment or Sequentialassignment1. COSY – Cross peak ‡ unique for each aminoacid, “fingerprint” Which a.a. in sequence?2. NOESYa. interactions in spaceb. interactions of residues that are sequentiallyadjacent (i and i+1)3. Amino Acid Sequence4. Distance Constraints: for H atoms in i to Hatoms in j{Cross-peaksInterpretation cont…5. Structure Refinements- computer modeling- No unique structure but differentstructures that are compatible with data(ambiguity)By Combination ofx-ray and NMR• Structure Determination• Complementary• In general agreement (except minordiscrepaencies especially loops)• Both require:Biochemical InformationSupercomputer
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