1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Thomas Blicher, Center for Biological Sequence Analysis Details of Protein Structure Folds, evolution & experimental methods

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Learning Objectives  Outline the basis for rational protein design.  Outline key differences between X-ray crystallography and NMR spectroscopy.  Identify relevant parameters for evaluating the quality of protein structures determined by X-ray crystallography and NMR spectroscopy.

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Outline  Protein structure evolution, function and folds  Inferring function from structure.  Modifying function  Experimental techniques  X-ray crystallography  NMR spectroscopy  Structure validation

4. 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…

5. 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?

6. 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

7. 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

8. 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

9. 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…

10. 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.     

11. 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.

12. 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.

13. 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.

14. 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)

15. 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

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Major Classes in SCOP  Classes  All alpha proteins (  )  All beta (  )  Alpha and beta proteins (  /  )  Alpha and beta proteins (  +  )  Multi-domain proteins  Membrane and cell surface proteins  Small proteins    Hemoglobin (1BAB) Immunoglobulin (8FAB) Triose phosphate isomerase (1HTI) Lysozyme (1JSF)

17. 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. 2007 X-ray13116 30860 39706 NMR 2451 5368 6862 Other 338 200 250 Total15905 36428 46818

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Growth in the Number of Folds

19. 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

20. 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

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Grouping 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

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

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

24. 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

25. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Experimental Methods Crystallography & NMR spectroscopy

26. 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

27. 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.

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

29. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU The Importance of Resolution high low 4 Å 2 Å 3 Å 1 Å

30. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Key Parameters  Resolution  R values  Agreement between data and model.  Usually between 0.15 and 0.25, should not exceed 0.30.  B factors  Contributions from static and dynamic disorder  Well determined ~10-20 Å 2, intermediate ~20-30 Å 2, flexible 30- 50 Å 2, invisible >60 Å 2.  No. of observations vs. parameters  Ramachandran plot

31. 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.

32. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU NMR Basics  NMR is nuclear magnetic resonance  NMR spectroscopy is done on proteins IN SOLUTION  Only atoms 1 H, 13 C, 15 N (and 31 P) can be detected in NMR experiments  Proteins up to 30 kDa  Proteins stable at high concentration (0.5-1mM), preferably at room temperature

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

34. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Well-defined structures RMSDs < 0.6 Å Evalutation of NMR Structures  What is the atomic backbone RMSD? Less well-defined structures RMSDs > 0.6 Å 3GF1, Cooke et al. Biochemistry, 19911T1H, Andersen et al. JBC, 2004

35. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Evaluation of NMR Structures  What types of constraints?  Numbers of constraints? Distance restraints Angle restraints RDCs Hydrogen bonds In well-defined regions each residues have at least 20 restraints Best if no violations of distance restraints <0.5Å

36. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Evaluation of NMR Structures What regions in the structure are most well-defined? Look at the pdb ensembles to see which regions are well-defined 1RJH Nielbo et al, Biochemistry, 2003

37. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Which Structural Model?  Normally NMR structure models are listed according to the total energy and the number of violations.  Model 1 in the PDB file is often the one with lowest energy and fewest violations.  Use that model as template for modelling.

38. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU NMR versus X-ray Crystallography  Hydrogen atoms are observed!  Only 13 C, 15 N and 1 H are observed  Study of proteins in solution  Only proteins up to 30-40 kDa  No total “map” of the structure  Information used is incomplete and used as restraints  An ensemble of structures is submitted to PDB  The solved structure can be used for further dynamics characterization with NMR

39. 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!

40. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Summary  In evolution structure is conserved longer than both function and sequence.  X-ray crystallography  Proteins in crystal lattice  Many details – one model  Resolution, R-values, Ramchandran plot  NMR spectroscopy  Proteins in solution  Fewer details – many models  Violations, RMSD, Ramachandran plot

41. 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