1. Basics of protein structure and modeling Rui Alves

2. MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEAGVT KRPRFLFEIAMALNCDPVWLQYGTKRGKAA atgcaaactctttctgaacgcctcaagaagaggcgaattgcgttaaaaatgacgcaaaccgaa ctggcaaccaaagccggtgttaaacagcaatcaattcaactgattgaagctggagtaaccaa gcgaccgcgcttcttgtttgagattgctatggcgcttaactgtgatccggtttggttacagtacgg aactaaacgcggtaaagccgcttaa augcaaacucuuucugaacgccucaagaagaggcgaauugcguuaaaaaugacgcaaacc gaacuggcaaccaaagccgguguuaaacagcaaucaauucaacugauugaagcuggagua accaagcgaccgcgcuucuuguuugagauugcuauggcgcuuaacugugauccgguuug guuacaguacggaacuaaacgcgguaaagccgcuuaa Proteins are the primary functional manifestation of genomes DNA sequence RNA sequence protein sequence protein structure Protein function transcription translation Being able to predict the protein sequence from the gene sequence allows us to predict structure, which in turn helps us understand how the protein does what it does

3. DNA sequence to protein sequence From protein sequence to secondary structure Protein tertiary structure Predicting protein structure Outline

4. Predicting protein sequence from DNA sequence Protein sequence can be predicted by translating the cDNA and using the genetic code.

5. Translating cDNA to protein ATGTCTCTTATATGA… MetSerLeuIle Ter No Gene!!!!!

6. Translating cDNA to Protein

7. Translating yeast mitochondrial cDNA into protein sequence ATGTCTCTTATATGA………SECIS sequence MetSerThrMetsCys MetSerLeuIleTer There is a Gene with a considerably different protein sequence from the one we would predict from the universal genetic code!!!!!

8. DNA sequence to protein sequence From protein sequence to secondary structure Protein tertiary structure Predicting protein structure Outline

9. The sequence of AAs is the primary structure of proteins Sequence determines structure Amino acids don’t fall neatly into classes How we casually speak of them can affect the way we think about their behavior. For example, if you think of Cys as a polar residue, you might be surprised to find it in the hydrophobic core of a protein unpaired to any other polar group. But this does happen. The properties of a residue type can also vary with conditions/environment Amino acids are the primary building blocks of proteins

10. Grouping the amino acids by properties Livingstone & Barton, CABIOS, 9, 745-756, 1993.

11. Proteins are made by controlled polymerization of amino acids H 2 NCHC R 1 OH O H 2 NCHC R 2 OH O H 2 NCHC R 1 NH O CHC R 2 OH O peptide bond is formed + HOH residue 1 residue 2 two amino acids condense to form......a dipeptide. If there are more it becomes a polypeptide. Short polypeptide chains are usually called peptides while longer ones are called proteins. water is eliminated N or amino terminus C or carboxy terminus

12. Repeating torsion angles  /  angles characterize the secondary structure

13. Secondary structure elements in proteins beta-strand (nonlocal interactions) alpha-helix (local interactions) A secondary structure element is a contiguous region of a protein sequence characterized by a repeating pattern of main-chain hydrogen bonds and backbone phi/psi angles Reflect the tendency of backbone to hydrogen bond with itself in a semi-ordered fashion when compacted

14. Principal types of secondary structure found in proteins Repeating (f,y) values -63 o -42 o -57 o -30 o -119 o +113 o -139 o +135 o   -helix (1  5) (right-handed) 3 10 helix (1  4) Parallel  -sheet Antiparallel  -sheet

15. The alpha-helix: repeating i,i+4 h-bonds 2 1 3 4 5 7 8 9 6 10 11 12 By DSSP definitions, which of residues 1-12 are in the helix? Does this coincide with the residues in the helical region of phi-psi space? right-handed helical region of phi-psi space hydrogen bond -63 o -42 o   -helix (1  5) (right-handed) -60 -120 -180 0 60 120 180 -180 -120-60 060120

16.  strands/sheets Is this a parallel or anti-parallel sheet? 49 50 51 52 53 54 57 56 beta-strand region of phi- psi space By DSSP definitions, which of res 49-57 are in the sheet? Does this coincide with the residues in the beta-strand region of phi-psi space? -119 o +113 o  Parallel  -sheet -60 -120 -180 0 60 120 180 -180-120-60 060120180

17. Contact maps of protein structures 1avg--structure of triabin map of C  -C  distances < 6 Å rainbow ribbon diagram blue to red: N to C -both axes are the sequence of the protein near diagonal: local contacts in the sequence off-diagonal: long-range (nonlocal) contacts

18. If, from the primary structure one can predict secondary structure, then this may help in predicting protein function, via evolutionary relationships with known folds What does secondary structure teach

19. DNA sequence to protein sequence From protein sequence to secondary structure Protein tertiary structure Predicting protein structure Outline

20. Tertiary structure in proteins Single polypeptide chain The number and order of secondary structures in the sequence (connectivity) and their arrangement in space defines a protein’s fold or topology Pattern of contacts between side chains/backbone also an aspect of tertiary structure Outer surface and interior

21. Obvious interactions in native protein structures disulfide crosslinks polar interactions (hydrogen bond/salt bridge) hydrophobic interactions

22. The protein databank The protein databank is a central repository of protein structures http://www.rcsb.org/pdb/home/home.do

23. Major structure classification systems SCOP (Structural Classification of Proteins) CATH (Class-Architecture-Topology-Homology) DALI/FSSP (Fold classification based on Structure- Structure Alignment) SCOP and CATH are quite similar and generally combine automated and manual aspects. They are both “curated” by human experts.

24. DNA sequence to protein sequence From protein sequence to secondary structure Protein tertiary structure Predicting protein structure Outline

25. Training set of known structures Training set of corresponding sequences Test set of known structures Test set of corresponding sequences The knuts and bolts behind fold predition p(  -helix) p(coil) p(  -strand) A0.230.280.5 Database of known structures Database of corresponding sequences ACDEFGTYAEE… …  -helix coil  -strand p(  -helix) p(coil) p(  -strand) A…C…A…C..A…C… A0.1…0.030.04…0.0020.1…0.21 p(aa1-coil) p(aa1-helix) p(aa1-strand) … Predict 2 ary structure Compare Bad Predictions: Reshuffle training set and test set and repeat until predictions are correct Good Predictions: Method ready for new sequence 2 ndary structure prediction

26. How does a fold prediction server work? Database of known structures Database of corresponding sequences Database of probabilities of aa in 2 ndary structure YOUR SEQUENCE Homology based helix coil-strand profile folds database Server Strong Homology … Fold Prediction Weak/No Homology Helix-coil-strand profile prediction … Fold Prediction

27. Predicting protein folding

28. Predicting protein structure Homology Modeling –3D-JIGSAW, SWISSMODEL Ab initio Modeling –ROBETTA

29. Predicting protein structure by homology

30. How does a homology modeling server work? Database of known structures Database of corresponding sequences …YDVRSEQVENCE… Server/ Program Strong Homologues Best possible alignment (Sequence+ Structure) …YDVR-SEQVENCE… …YDVRMSD-VDNCD… …YDVR-SEQVENCE… …YDVRMSD-VDNCD… … … Thread sequence to predict over known structure according to alignment … … Optimization via energy minimization, etc…

31. Predicting protein structure Homology Modeling –3D-JIGSAW,SWISSMODEL Ab initio Modeling –ROSETTA

32. Predicting protein structure by ab initio methods Database of corresponding sequences …YDVRSEQVENCE… Server/ Program NO Homologues Database of structures for smaller amino acid runs …YDVR-SEQ …YDVRMSD-… …YDVR-SEQ …YPVRMSD-… … …VENCE… …YDNCD… …VENCE… …VEQCE… … … Assemble Energy minimization & optimization …

33. Accuracy of modelling Accuracy is widely varying. The quality of the model is VERY dependent on the quality of the alignment Globular proteins are more accurately predicted Membrane proteins are still a big problem Homology modelling is “bad” if Homology<30% CASP is a bienial meeting where accuracy of the different methods is predicted –Baker group is usually and consistently more accurate than others http://www.predictioncenter.org/

34. DNA sequence to protein sequence From protein sequence to secondary structure Protein tertiary structure Predicting protein structure Summary

35. “Accessible Surface” Lee & Richards, 1971 Shrake & Rupley, 1973 represent atoms as spheres w/appropriate radii and eliminate overlapping parts... mathematically roll a sphere all around that surface... the sphere’s center traces out a surface as it rolls...

36. The outer surface: water in protein structures Structures of water-soluble proteins determined at reasonably high resolution will be decorated on their outer surfaces with water molecules (cyan balls) with relatively well- defined positions, and waters may also occur internally Water is not just surrounding the protein--it is interacting with it

37. Water interacts with protein surfaces second shell water: only contacts other waters first shell waters: in contact with/ hydrogen bound to protein most waters visible in structures make hydrogen bonds to each other and/or to the protein, as donor/acceptor/both

38. Side chain conformation side chains differ in their number of degrees of conformational freedom (some don’t have any, such as Ala and Gly) but side chains of very different size can have the same number of c angles.

39. Supersecondary structures/structural motifs just as there are certain secondary structure elements that are common, there are also particular arrangements of multiple secondary structure elements that are common supersecondary structures emphasize issue of topology in protein structure  motif greek key motif

40. Topology: differences in connectivity “greek key”“up-and-down” example: a four-stranded antiparallel b sheet can have many different topologies based on the order in which the four b strands are connected:

41. Topology: differences in handedness example: An extremely common supersecondary structure in proteins is the beta-alpha-beta motif, in which two adjacent beta-strands are arranged in parallel and are separated in the sequence by a helix which packs against them. if the two parallel strands are oriented to face toward you, the helix can be either above or below the plane of the strands. huge preference for right-handed arrangement in proteins

42. DIY: The sequence

43. DIY: The server

44. DIY: The reply

45. DIY: fine tuning

46. DIY: That is it!

47. The CATH Hierarchy 1. Divide PDB structure entries into domains (using domain recognition algorithms--domain is the fundamental unit of structure classification 2. Classify each domain according to a five level hierarchy: Class Architecture Topology Homologous Superfamily Sequence Family the top 3 levels of the hierarchy are purely phenetic--based on characteristics of the structure, not on evolutionary relationships the bottom two levels include some phyletic classification as well-- groupings according to putative common ancestry based on structural similarity, functional similarity, and sequence similarity There is no purely phyletic system of protein classification! (also unlikely that there is any common ancestor to all proteins)

48. SCOP: A different (but similar) taxonomy system Correspondences between SCOP and CATH hierarchies: SCOPCATHclass architecture foldtopology homologous superfamily superfamily familysequence familydomain CATH more directed toward structural classification, whereas SCOP pays more attention to evolutionary relationships. Both have in common that they have manual aspects and are curated by experts.

49. Internal interactions in a protein

50. Amino acids: the building blocks of proteins H 2 NCHC R OH O H 3 NCHC R O O The zwitterionic form is the predominant form at neutral pH amino group carboxylic acid group side chain alpha carbon H 3 N C C R O O H