1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Protein Structure Thomas Blicher, Center for Biological Sequence Analysis

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU “Could the search for ultimate truth really have revealed so hideous and visceral-looking an object?” Max Perutz, 1964, on protein structure John Kendrew, 1959, with myoglobin model Once Upon a Time…

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Holdings of the Protein Data Bank (PDB): The PDB also contains nucleotide and nucleotide analogue structures. PDB Sep. 2001 May 2006 Oct. 2006 X-ray13116 30860 33433 NMR 2451 5368 5810 Other 338 200 221 Total15905 36428 39464

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  X-ray crystallography  Nuclear Magnetic Resonance (NMR)  Modelling techniques  More exotic techniques  Cryo electron microscopy (Cryo EM)  Small angle X-ray scattering (SAXS)  Neutron scattering Methods for Structure Determination

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU X-ray Crystallography  No size limitation.  Protein molecules are ”stuck” in a crystal lattice.  Some proteins seem to be uncrystallizable.  Slow.  Especially suited for studying structural details.

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU X-rays Fourier transform

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU NMR Spectroscopy  Upper limit for structure determination currently ~50 kDa.  Protein molecules are in solution.  Dynamics, protein folding.  Slow.  Especially suited for studies of protein dynamics of small to medium size proteins.

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU NMR Spectroscopy

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Modelling  Need structure of a >30% id homolog.  Only applicable to ~50% of sequences.  Fast.   Accuracy poor for low sequence id.  There is still need for experimental structure determination!

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Amino Acids  Amino group and acid group  Side chain at C   Chiral, only one enantiomer found in proteins (L-amino acids) N O C Ca

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU http://www.ch.cam.ac.uk/magnus/molecules/amino/

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Amino Acids Livingstone & Barton, CABIOS, 9, 745-756, 1993 A – Ala C – Cys D – Asp E – Glu F – Phe G – Gly H – His I – Ile K – Lys L – Leu M – Met N – Asn P – Pro Q – Gln R – Arg S – Ser T – Thr V – Val W – Trp Y - Tyr

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU The Evolution Way  Based on Blosum62 matrix  Measure of evolutionary substitution probability

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Structure Levels  Primary structure = Sequence  Secondary Structure = Helix, sheets/strands, loops & turns  Structural Motif = Small, recurrent arrangement of secondary structure, e.g.  Helix-loop-helix  Beta hairpins  EF hand (calcium binding motif)  Etc.  Tertiary structure = Arrangement of Secondary structure elements MSSVLLGHIKKLEMGHS…

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Proteins Are Polypeptides  The peptide bond  A polypeptide chain

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Ramachandran Plot  Allowed backbone torsion angles in proteins N H

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Small Exercise (3 minutes)  For the polypeptide on the left discuss the following with your neighbour:  Why is the lower left quadrant a ”forbidden” region in the Ramachandran plot?  What makes Gly a special amino acid when it comes to Ramachandran plots?  What about Pro?

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Hydrophobic Core  Hydrophobic side chains go into the core of the molecule – but the main chain is highly polar.  The polar groups (C=O and NH) are neutralized through formation of H-bonds. Myoglobin SurfaceInterior

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Secondary Structure  Two main types:  Helices (mainly  -helix)  Sheets (consisting of individual strands)  Other types of secondary structure:  3 10 helices (C=O (n) … HN (n+3) )   -helices (C=O (n) … HN (n+5) )   -turns and loops (in old textbooks sometimes referred to as random coil)

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Characteristics of Helices  Aligned peptide units  Dipolar moment  Ion/ligand binding  Secondary and quaternary structure packing  Capping residues  The  helix (i→i+4)  Other helix types! (3 10,  ) N C

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  -Sheets  Multiple strands  sheet  Parallel vs. antiparallel  Twist  Flexibility  Vs. helices  Folding  Structure propagation (amyloids)  Other… Thioredoxin

22. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  -Sheets  Multiple strands  sheet  Parallel vs. antiparallel  Twist  Flexibility  Vs. helices  Folding  Structure propagation (amyloids)  Other…

23. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  -Sheets  Multiple strands  sheet  Parallel vs. antiparallel  Twist  Flexibility  Vs. helices  Folding  Structure propagation (amyloids)  Other…

24. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  -Sheets  Multiple strands  sheet  Parallel vs. antiparallel  Twist  Flexibility  Vs. helices  Folding  Structure propagation (amyloids)  Other…

25. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Two Types of  -Sheet

26. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Two Types of  -Sheet  Anti-parallel  Parallel

27. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Rhamnogalacturonan lyase (1nkg) Rhamnogalacturonan acetylesterase (1k7c) Tertiary Structure  Domains and modules

28. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  Myoglobin  Hemoglobin Quaternary Structure  Assembly of monomers/subunits into protein complex  Backbone-backbone, backbone-side-chain & side-chain-side-chain interactions:  Intramolecular vs. intermolecular contacts.  For ligand binding side chains may or may not contribute. For the latter, mutations have little effect.     

29. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Classification Schemes  SCOP  Manual classification (A. Murzin)  CATH  Semi manual classification (C. Orengo)  FSSP  Automatic classification (L. Holm)

30. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Levels in SCOP Class# Folds# Superfamilies # Families All alpha proteins202342550 All beta proteins141280529 Alpha and beta proteins (a/b)130213593 Alpha and beta proteins (a+b)260386650 Multi-domain proteins404055 Membrane and cell surface proteins428291 Small proteins72104162 Total88714472630 http://scop.berkeley.edu/count.html#scop-1.67

31. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Major Classes in SCOP  Classes  All alpha proteins  Alpha and beta proteins (  /  )  Alpha and beta proteins (  +  )  Multi-domain proteins  Membrane and cell surface proteins  Small proteins

32. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU All   Hemoglobin (1BAB)

33. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU All   Immunoglobulin (8FAB)

34. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU   Triose phosphate isomerase (1HTI)

35. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU   Lysozyme (1JSF)

36. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Folds*  Proteins which have >~50% of their secondary structure elements arranged the in the same order in the protein chain and in three dimensions are classified as having the same fold.  No evolutionary relation between proteins. *confusingly also called fold classes.

37. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Superfamilies  Proteins with (remote) evolutionary relations  Sequence similarity low  Share function  Share special structural features  Relationships between members of a superfamily may not be readily recognizable from the sequence alone.

38. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Families  Proteins whose evolutionarily relationship is readily recognizable from the sequence (>~25% sequence identity).  Families are further subdivided into Proteins.  Proteins are divided into Species  The same protein may be found in several species.

39. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Links  PDB (protein structure database)  www.rcsb.org/pdb/ www.rcsb.org/pdb/  SCOP (protein classification database)  scop.berkeley.edu scop.berkeley.edu  CATH (protein classification database)  www.biochem.ucl.ac.uk/bsm/cath www.biochem.ucl.ac.uk/bsm/cath  FSSP (protein classification database)  www.ebi.ac.uk/dali/fssp/fssp.html www.ebi.ac.uk/dali/fssp/fssp.html

40. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  They provide a detailed picture of interesting biological features, such as active site, substrate specificity, allosteric regulation etc.  They aid in rational drug design and protein engineering.  They can elucidate evolutionary relationships undetectable by sequence comparisons. Why are Protein Structures so Interesting?

41. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU COOH NH 2 Asp His Ser Topological switchpoint Inferring biological features from the structure 1DEO Structure to Function

42. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Active site Triose phosephate isomerase (1AG1) (Verlinde et al. (1991) Eur.J.Biochem. 198, 53) Structure to Function  Inferring biological features from the structure

43. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Im, Ryu & Yu (2004) Engineering thermostability in serine protease inhibitors PEDS, 17, 325-331. Engineering Thermostability Example: Serpin (serine protease inhibitor)  Overpacking  Buried polar groups  Cavities

44. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  In evolution structure is conserved longer than both function and sequence. Structure > Function > Sequence Structure & Evolution

45. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Rhamnogalacturonan acetylesterase (A. aculeatus) (1k7c) Platelet activating factor acetylhydrolase (B. Taurus) (1WAB) Serine esterase (S. scabies) (1ESC) Structure & Evolution

46. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Platelet activating factor acetylhydrolase Serine esterase Rhamnogalacturonan acetylesterase Mølgaard, Kauppinen & Larsen (2000) Structure, 8, 373-383. Structure & Evolution

47. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Quote of the Day  Don C. Wiley (1944-2001)  Has solved the structures of many immunologically important proteins:  Class I and II Major Histocompatibility Complex proteins.  Has studied viruses including HIV, Herpes and Influenza virus and many more.  Special focus on molecules involved in viral entry. “I’m sorry, but I just don’t understand anything in biology unless I know what it looks like.”