1. Structure-Function Analysis 117 Jan 2006 DNA/Protein structure-function analysis and prediction Protein Structure Determination: –X-ray Diffraction (Titia Sixma, NKI)‏ –Electron Microscopy/Diffraction (Titia Sixma, NKI)‏ –NMR Spectroscopy (Lorna Smith, Oxford)‏ –Other Spectroscopic methods

2. Structure-Function Analysis 217 Jan 2006 Spectroscopy: The whole spectrum

3. Structure-Function Analysis 317 Jan 2006 DNA/Protein structure-function analysis and prediction Protein Structure Determination: –X-ray Diffraction –Electron Microscopy/Diffraction –NMR Spectroscopy –Other Spectroscopic methods

4. Structure-Function Analysis 417 Jan 2006 Structure determination method X-ray crystallography Purified protein Crystal X-ray Diffraction Electron density 3D structure Biological interpretation Crystallization Phase problem

5. Structure-Function Analysis 517 Jan 2006 Protein crystals Regular arrays of protein molecules ‘Wet’: 20-80% solvent Few crystal contacts Protein crystals contain active protein Enzyme turnover Ligand binding Example of crystal packing

6. Structure-Function Analysis 617 Jan 2006 Examples of crystal packing  2 Glycoprotein I ~90% solvent (extremely high!)‏ Acetylcholinesterase ~68% solvent

7. Structure-Function Analysis 717 Jan 2006 Problematic proteins Multiple domains Similarly, floppy ends may hamper crystallization: change construct Membrane proteins Glycoproteins Flexible Lipid bilayer hydrophilic hydrophobic Flexible and heterogeneous!!

8. Structure-Function Analysis 817 Jan 2006 Experimental set-up Options for wavelength: –monochromatic, polychromatic –variable wavelength Liq.N 2 gas stream X-ray source detector goniometer beam stop

9. Structure-Function Analysis 917 Jan 2006 Diffraction image Water ring Diffuse scattering (from the fibre loop)‏ reciprocal lattice (this case hexagonal)‏ Beam stop Increasing resolution Direct beam Reflections (h,k,l) with I(h,k,l)‏

10. Structure-Function Analysis 1017 Jan 2006 The rules for diffraction: Bragg’s law Scattered X-rays reinforce each other only when Bragg’s law holds: Bragg’s law: 2d hkl sin  = n

11. Structure-Function Analysis 1117 Jan 2006 Phase Problem If phases  hkl and structure factor F(hkl) known: compute the electron density  (x,y,z)‏ –In the electron density build the atomic 3D model However, the phases  hkl are unknown !

12. Structure-Function Analysis 1217 Jan 2006 F H,  H F K,  K F H,  K F K,  H How important are these phases ?? Fourier transform photo’s of Karle (top left) and Hauptman (top right) (two crystallography pioneers)‏ Combine amplitudes F K with phase  H and inverse-fourier transform Combine amplitudes F H with phase  K and inverse-fourier transform (Taken from: Randy J. Read )‏

13. Structure-Function Analysis 1317 Jan 2006 How can we solve the Phase Problem ? Direct Methods –small molecules and small proteins –needs atomic resolution data ( d < 1.2 Å) ! Difference method using heavy atoms –multiple isomorphous replacement (MIR)‏ –anomalous scattering (AS)‏ –combinations (SIRAS,MIRAS) Difference method using variable wavelength –multiple-wavelength anomalous diffraction (MAD)‏ Using a homologous structure –molecular replacement

14. Structure-Function Analysis 1417 Jan 2006 Building a protein model Find structural elements:  -helices,  -strands Fit amino-acid sequence

15. Structure-Function Analysis 1517 Jan 2006 Building a protein model Find structural elements:  -helices,  -strands Fit amino-acid sequence

16. Structure-Function Analysis 1617 Jan 2006 Effects of resolution on electron density Note: map calculated with perfect phases d = 4 Å

17. Structure-Function Analysis 1717 Jan 2006 d = 3 Å Effects of resolution on electron density Note: map calculated with perfect phases

18. Structure-Function Analysis 1817 Jan 2006 d = 2 Å Effects of resolution on electron density Note: map calculated with perfect phases

19. Structure-Function Analysis 1917 Jan 2006 d = 1 Å Effects of resolution on electron density Note: map calculated with perfect phases

20. Structure-Function Analysis 2017 Jan 2006 Refinement process Bad phases  poor electron density map  errors in the protein model Interpretation of the electron density map  improved model  improved phases  improved map  even better model … iterative process of refinement

21. Structure-Function Analysis 2117 Jan 2006 Validation Free R-factor (cross validation)‏ –Number of parameters/ observations Ramachandran plot Chemically likely (WhatCheck)‏ –Hydrophobic inside, hydrophilic outside –Binding sites of ligands, metals, ions –Hydrogen-bonds satisfied –Chemistry in order Final B-factor values

22. Structure-Function Analysis 2217 Jan 2006 DNA/Protein structure-function analysis and prediction Protein Structure Determination: –X-ray Diffraction –Electron Microscopy/Diffraction –NMR Spectroscopy –Other Spectroscopic methods

23. Structure-Function Analysis 2317 Jan 2006 Electron microscopy Single particle –Low resolution, not really atomic –Less purity of protein, more transient state analysis Two-dimensional crystals –Suited to membrane proteins Fibres –Acetylcholine receptor –Muscles, kinesins and tubulin Preserve protein by –Negative stain (envelope only)‏ –Freezing in vitreous ice (Cryo-EM, true density maps)‏ High resolution possible but difficult to achieve –For large complexes: Combine with X-ray models

24. Structure-Function Analysis 2417 Jan 2006 Electron diffraction from 2D crystals: Nicotinic Acetylcholine Receptor Unwin et al, 2005 G-protein coupled receptors

25. Structure-Function Analysis 2517 Jan 2006 Electron diffraction: near atomic resolution Structure of the alpha beta tubulin dimer by electron crystallography. Nogales E, Wolf SG, Downing KH. Nature 1998 391 199-203

26. Structure-Function Analysis 2617 Jan 2006 Select particlesSort into classes AverageReconstruct 3D image Single particle Electron Microscopy

27. Structure-Function Analysis 2717 Jan 2006 Leiman et al. Cell. 2004 Aug 20;118(4):419-29 Cryo EM reconstruction: Tail of bacteriophage T4

28. Structure-Function Analysis 2817 Jan 2006 DNA/Protein structure-function analysis and prediction Protein Structure Determination: –X-ray Diffraction –Electron Microscopy/Diffraction –NMR Spectroscopy –Other Spectroscopic methods

29. Structure-Function Analysis 2917 Jan 2006 1D NMR spectrum of hen lysozyme (129 residues)‏ Too much overlap in 1D:  2D 1 H- 1 H 1 H- 15 N 1 H- 13 C

30. Structure-Function Analysis 3017 Jan 2006 Val  Val  Thr  Thr  2D COSY spectrum of peptide in D 2 O

31. Structure-Function Analysis 3117 Jan 2006 Nitroreductase Dimer: 217 residues Too much overlap in 2D:  3D 1 H- 1 H- 15 N 1 H- 13 C- 15 N

32. Structure-Function Analysis 3217 Jan 2006 Structural information from NMR: NOEs For macromolecules such as proteins: –Initial build up of NOE intensity  1/r 6 –Between protons that are < 5Å apart

33. Structure-Function Analysis 3317 Jan 2006 Structural information from NMR 3. Hydrogen exchange rates Dissolve protein in D 2 O and record series of spectra –Backbone NH ( 1 H) exchange with water ( 2 H)‏ Slow exchange for NH groups –In hydrogen bonds –Buried in core of protein After 20 minsAfter 68 hours 1 H- 15 N HSQC spectra for SPH15 in D 2 O

34. Structure-Function Analysis 3417 Jan 2006 NMR structure determination: hen lysozyme 129 residues –~1000 heavy atoms –~800 protons NMR data set –1632 distance restraints –110 torsion restraints –60 H-bond restraints 80 structures calculated 30 low energy structures used 0 2000 4000 6000 8000 1 10 4 1.2 10 4 10203040506070 Total energy Structure number

35. Structure-Function Analysis 3517 Jan 2006 Solution Structure Ensemble Disorder in NMR ensemble –lack of data ? –or protein dynamics ?

36. Structure-Function Analysis 3617 Jan 2006 Summary Resolution variable Limited size (< ~30kDa)‏ In solution Dynamic information possible NMR spectroscopy Labour intensive High resolution difficult Single particles possible Well suited to membrane proteins Electron microscopy / diffraction Technically extremely complex Limited applicability Limited information (Very) high time resolution (ps!)‏ Very sensitive Other spectroscopy Crystals required Phase problem High resolution No size limits Easy addition of ligands X-ray crystallography ConProMethod

37. Structure-Function Analysis 3717 Jan 2006 Supplementary Slides The following slides were not covered in the lectures, and will not be part of the examination material. They are provided for students with one or both of the following characteristics: –that unsatisifyable hunger for knowledge: “I simply need to know this, my life will not be complete without!” –that urge to be meticulously precise: “I really cannot stand that anything could be left out, please put it back in!” For your information, there is about 25 years worth of study left out of this lecture ;-)‏

38. Structure-Function Analysis 3817 Jan 2006 N C  C N C  C H O N H O H C  H RH H H C  Amino acid spin system C O Step 1: Identification of amino acid spin systems

39. Structure-Function Analysis 3917 Jan 2006 N C  C N C  C H O N H O H  C  H RH H H C  NH(i)-NH(i+1)‏ NOE  H(i)-NH(i+1) NOE  NH(i)-NH(i+1)‏ NOE Step 2: Sequential assignment

40. Structure-Function Analysis 4017 Jan 2006 Gly Asp Asn Asp Phe Thr Ser Leu Val 2D NOESY spectrum Peptide sequence (N-terminal NH not observed)‏ Arg-Gly-Asp-Val-Asn-Ser-Leu-Phe-Asp-Thr-Gly

41. Structure-Function Analysis 4117 Jan 2006 NOEs in  -helices NH-NH(i,i+1)2.8Å  H-NH(i,i+3)3.4Å  H-  H(i,i+3)2.5-4.4Å Cytochrome c 552 3D 1 H- 15 N NOESY –  H-NH(i,i+3)‏ –resides 38-47 –form  -helix

42. Structure-Function Analysis 4217 Jan 2006 NOEs in  -strands  H-NH(i,j) 2.3Å  H-NH(i,j) 3.2Å

43. Structure-Function Analysis 4317 Jan 2006 Long(er) range NOEs Provide information about –Packing of amino acid side chains –Fold of the protein NOEs observed for C  H of Trp 28 in hen lysozyme

44. Structure-Function Analysis 4417 Jan 2006 N C  C N C  C H O N H O H C  H RH H H C  C O   Spin-spin coupling constants Fine structure in COSY cross peaks For proteins 3J(HN.H  ) useful: –Probes main chain  torsion angle Hen lysozyme: 2 3 4 5 6 7 8 9 10 020406080100 120 140 Coupling constant (Hz)‏ Residue number

45. Structure-Function Analysis 4517 Jan 2006 Structural information from NMR NOEs –1H  - 2H  1.8-2.5Å(strong)‏ –2H  - 40HN1.8-5.0Å(weak)‏ –3H  - 88H  21.8-5.0Å(weak)‏ –3H  - 55H  1.8-3.5Å(medium)‏ Spin-spin coupling constants –1C-2N-2C  -2C-120+/-40° –4C-5N-5C  -5C-60+/- 30° –5C-6N-6C  -6C-60+/- 30° Hydrogen exchange rates –10HN-6CO1.3-2.3Å10N-6CO2.3-3.3Å –11HN-7CO1.3-2.3Å11N-7CO2.3-3.3Å

46. Structure-Function Analysis 4617 Jan 2006 DNA/Protein structure-function analysis and prediction Protein Structure Determination: –X-ray Diffraction –Electron Microscopy/Diffraction –NMR Spectroscopy –Other Spectroscopic methods

47. Structure-Function Analysis 4717 Jan 2006 Ultrafast Protein Spectroscopy Structure-sensitive technique State of protein and substrate –Redox state –Protonation –Elektronen Follow reactions in real-time Why ultrafast spectroscopy? –Molecular movement: time scales of 10 fs – 1 ps (10 -15 – 10 -12 s)‏ –O  H stretching frequency: ~3500 cm -1 (~ 10 -14 s)‏ Transfer or movement of proton, electron or C-atom

48. Structure-Function Analysis 4817 Jan 2006 keto ester Keto and ester C=O in Chlorophyll a, Pheophytin a are redox and environmental probes Excited state difference spectra of Chl and Pheo

49. Structure-Function Analysis 4917 Jan 2006 X – C – O – H O – X O ω1ω1 ω2ω2 ω1’ω1’ ω2’ω2’ Why is vibrational spectroscopy sensitive to structure?

50. Structure-Function Analysis 5017 Jan 2006 H-bond response during the photocycle Replace Glu with Gln which donates weaker H-bond weakerstronger broken