1. Protein structure and modelling ● Orientation ● Protein structure ● Protein modelling Andreas Heger University of Helsinki Bioinformatics Group Slides will be available at: ekhidna.biocenter.helsinki.fi:8080/downloads/teaching/hut2004/

2. Proteins ● Proteins are involved in all processes inside a cell – Gene regulation – Metabolism – Signalling – Development – Structure http://www.websters-online-dictionary.org/definition/english/ce/cell.html

3. Chemistry ● Proteins are linear hetero-polymers of amino acids – twenty different amino acids (building blocks) ARG LYS VAL ILE PRO ARG GLU LYS R K V I P R E K 3-letter code 1-letter code

4. Peptide bond http://www.imb-jena.de/~rake/Bioinformatics_WEB/basics_peptide_bond.html The peptide bond is planar 2 angles freely rotatable 1 is fixed Peptide ~ 2-10 amino acids Polypeptide ~ 10-50 amino acids Protein~ 50- amino acids Double bond character of the peptide bond

5. Amino acids ● Side chain properties – Size – Charge – Polarity http://www.ch.cam.ac.uk/SGTL/Structures/amino/

6. Proteins are very special polymers: ● A given protein has always the same amino acid sequence – Protein sequence is determined by DNA sequence ● A given protein has always a unique three- dimensional structure. – Protein structure is determined by protein sequence. always = biological always (there are exceptions)

7. Protein evolution Sequence – Structure - Function DNA sequence Protein sequence Protein structure Protein function Selection

8. Summary ● Protein structure is the key to understanding protein function ● Topics in protein structure 1.Protein structure determination 2.Protein architecture 3.Protein function 4.Protein folding ● Protein modelling and computational methods

9. Protein structure determination ● Protein expression – membrane proteins – aggregation ● X-Ray crystallography ● NMR (nuclear magnetic resonance) ● Cryo-EM (electron microscopy)

10. Structures by X-ray crystallography ➔ Crystallize protein ● Collect diffraction patterns ● Improve iteratively: – Calculate electron density map ● Phase problem – Fit amino acid trace through map

11. X-ray crystallography ● Crystallization ● “An art as much as a science” Charges http://crystal.uah.edu/~carter/protein/crystal.htm

12. Diffraction and electron density maps Diffraction pattern X-ray source Crystal Intensities

13. Iterative refinement http://www.sci.sdsu.edu/TFrey/Bio750/Bio750X- Ray.html Higher resolution = more accurate positioning of atoms Resolution

14. NMR ● Create highly concentrated protein solution ● Record spectra ● Assign peaks to residues ● Calculate constraints ● Compute structure

15. NMR spectra 1D 2D http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/2dnmr.htm

16. Distance constraints from NMR ● From the sequence – Topology – Bond angles – Bond lengths ● From the NMR experiment – Torsion angles – Distance constraints HαHα R CO H Torsion angle

17. Ensemble of structures SH3-domain 1aey

18. What is the true protein structure? ● X-Ray – “frozen” state of a protein ● crystal contacts ✔ large protein structure ● NMR ✔ protein in solution – limited in size

19. Molecular complexes via X-ray 1fjg 30 S subunit of the ribosome Protein RNA

20. Cryo-EM Single particle image reconstruction Koning et al. (2003) Bacteriophage MS2

21. Fitting X-Ray structures into density maps

22. GroEL- complex 1gr6 Hemoglobin

23. Protein structure databases http://www.wwpdb.org/index.ht ml

24. Protein architecture ● Protein structure is the key to understanding protein function ● Topics in protein structure 1.Protein structure determination 2.Protein architecture 3.Protein function 4.Protein folding ● Protein modelling and computational methods

25. Topics in protein architecture ● Principles of protein architecture – Secondary structure – Supersecondary structure – Tertiary structure – Quarternary structure ● Classification of protein structures

26. The big surprise DNA is a regular structure Watson & Crick (1953)

27. Myoglobin Kendrew and Perutz 1957 1mbn

28. Secondary structure ● backbone – no amino acid side chains ● regular patterns – of hydrogen-bonds – backbone torsion angles ● types of secondary structure – α-helix – β-sheet –...

29. α-Helix β-Sheet hydrogen bond pattern: n, n+4

30. β-sheet http://broccoli.mfn.ki.se/pps_course_96 view from the top view from the side β-strands

31. Cartoon representation 2TRX 2AAC

32. Supersecondary structures ● local arrangments of secondary structure elements http://www.expasy.org/swissmod/course/text/chapter2.htm

33. Tertiary structure 1coh

34. Quaternary structure 1coh

35. Protein structure ● Primary structure ● Secondary structure ● Super-secondary structure ● Tertiary structure ● Quaternary structure

36. Protein domains/modules ● globular ● independently foldable ● occur in different contexts

37. Domains via the contact matrix

38. Structure classification ● 24908 structures in the Protein Databank (PDB) ● major classifications of proteins: – SCOP http://scop.mrc-lmb.cam.ac.uk/scop/ – CATH http://www.biochem.ucl.ac.uk/bsm/cath/ – DALI DOMAIN DICTIONARY/FSSP http://ekhidna.biocenter.helsinki.fi:8080/dali/index.html

39. Hierachical description of protein architecture 1.Class: α, β, α/β, α+β 2.Fold Structural similarity 3.Superfamily Evolutionary relationship 4.Family Sequence similarity 1.Class α, β, α&β 2.Architecture SS: Spatial arrangement 3.Topology SS: Topology 4.Homology structural/sequence similarity SCOP CATH

40. http://www.biochem.ucl.ac.uk/bsm/cath/cath_info.html Class Architecture Topology

41. Dali Domain Dictionary 1.Fold space attractor region Secondary structure composition and supersecondary structural motifs 2.Globular folding topology Structural comparison 3.Functional family Neural network 4.Sequence family Sequence comparison

43. Deviation from globularity ● Domain swapping ● Repetitive structures ● Open/closed conformations 1bsr 5rsa 1amy 1d0b

44. Protein function ● Protein structure is the key to understanding protein function ● Topics in protein structure 1.Protein structure determination 2.Protein architecture 3.Protein function 4.Protein folding ● Protein modelling and computational methods

45. Topics in protein function ● How does structure determine function? – Structural proteins – Enzymes – Transcription factors –...

46. Structural proteins ● Collagen 1K6Fhttp://www.aw-bc.com/mathews/ch06/fi6p13ad.htm

47. Actin and muscles

48. Enzymes ● Catalytic triad: Asp, Ser, His 1CHO

49. Mechanism ● Enzymes speed up chemical reactions ● Enzymes are not consumed by the reaction ● Stabilization of the transition state ● Charge-relay cascade

50. Convergent evolution in serine proteases ● same reaction ● same mechanism ● same orientation of catalytic residues ● different structures – Chymotrypsin: ● His-57, Asp-102, Ser-195 – Subtilisin: ● Asp-32, His-64, Ser-221 1cho / 1sib

51. Substrate specificity Perona & Craik (1997)

52. Transcription factors 1L3L Ligand DNA

53. Hydrogen bonding pattern Vannini (2002)

54. Protein folding ● Protein structure is the key to understanding protein function ● Topics in protein structure 1.Protein structure determination 2.Protein architecture 3.Protein function 4.Protein folding ● Protein modelling and computational methods

55. Protein denaturation ● Denatured state = unfolded state ● Native state = folded state ● Denaturation = heat, urea, salts Reaction coordinate Energy Folded Unfolded Reaction coordinate Energy Folded Unfolded

56. Protein stability ● Native state only marginally more stable than denatured state ● Contributions to protein stability – hydrophobic effect: entropic effect – hydrogen bonds: net effect = 0 – others ● salt bridges ● disulphide bonds ● aromatic-aromatic interactions ● metal binding

57. Hydrophobic core of lysozyme 1HEL Hydrophobic amino acid Hydrophilic amino acid

58. Protein folding ● Folding Funnel ● Energy landscape guides protein towards native structure Dobson (2004) C: total contacts Q: native contacts

59. Energy landscape for the folding of lysozyme Fast track Slow track Dobson (2004)

60. Misfolded proteins ● Disulfid-isomerases, Prolin-isomerases ● Chaperones: unfold misfolded proteins ● Protein folding diseases – BSE – Alzheimer's disease – Parkinson's disease –...

61. GroEL – a chaperone 1gr6

62. Wang & Weissmann (1999) Roseman et al. (1996) GroEL mechanism

63. Protein structure ● Protein structure is the key to understanding protein function ● Topics in protein structure 1.Protein structure determination 2.Protein architecture 3.Protein function 4.Protein folding ● Protein modelling and computational methods