1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Protein structure Anne Mølgaard, 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

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Holdings of the Protein Data Bank (PDB): Sep. 2001Feb. 2005 X-ray13116 25350 NMR24514383 theoretical3380 total1590529733

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Methods for structure determination X-ray crystallography Nuclear Magnetic Resonance (NMR) Modelling techniques

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

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

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Modeling 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!

9. 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 CC

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

11. 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

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Levels of protein structure: Primary Secondary Tertiary Quarternary

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Primary structure MKTAALAPLFFLPSALATTVYLA GDSTMAKNGGGSGTNGWGEYL ASYLSATVVNDAVAGRSAR… (etc)

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  -helix  -sheet left-handed  -helix Ramachandran plot

15. 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

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Secondary structure  -helix C=O (n) … HN (n+4)  -sheet (anti-parallel)

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU … and all the rest 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)

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU The  -helix has a dipole moment + - C N

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Two types of  -sheet: anti-parallel parallel

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Tertiary structure (domains, modules) Rhamnogalacturonan lyase (1nkg) Rhamnogalacturonan acetylesterase (1k7c)

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Quarternary structure B.caldolyticus UPRTase (1i5e) B.subtilis PRPP synthase (1dkr)

22. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU A. aculeatus RG acetylesterase Protein structure and water

23. 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)

24. 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

25. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Major classes in SCOP Classes –All alpha proteins –Alpha and beta proteins (a/b) –Alpha and beta proteins (a+b) –Multi-domain proteins –Membrane and cell surface proteins –Small proteins

26. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU All  : Hemoglobin (1bab)

27. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU All  : Immunoglobulin (8fab)

28. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  Triose phosphate isomerase (1hti)

29. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU  : Lysozyme (1jsf)

30. 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

31. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Superfamilies Proteins which are (remote) evolutionarily related –Sequence similarity low –Share function –Share special structural features Relationships between members of a superfamily may not be readily recognizable from the sequence alone

32. 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

33. 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.eduscop.berkeley.edu CATH (protein classification database) –www.biochem.ucl.ac.uk/bsm/cathwww.biochem.ucl.ac.uk/bsm/cath FSSP (protein classification database) –www.ebi.ac.uk/dali/fssp/fssp.htmlwww.ebi.ac.uk/dali/fssp/fssp.html

34. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Why are protein structures so interesting? 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

35. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU COOH NH 2 Asp His Ser Topological switchpoint Inferring biological features from the structure 1deo

36. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Inferring biological features from the structure Active site Triose phosephate isomerase (1ag1) (Verlinde et al. (1991) Eur.J.Biochem. 198, 53)

37. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Engineering thermostability in serpins Overpacking Buried polar groups Cavities Im, Ryu & Yu (2004) Engineering thermostability in serine protease inhibitors PEDS, 17, 325-331.

38. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Evolution... Structure is conserved longer than both sequence and function

39. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Rhamnogalacturonan acetylesterase (A. aculeatus) (1k7c) Platelet activating factor acetylhydrolase (Bos Taurus) (1wab) Serine esterase (S. scabies) (1esc)

40. 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.

41. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU "We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest…. …It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." J.D. Watson & F.H.C. Crick (1953) Nature, 171, 737.

42. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU