1. Protein analysis and proteomics (Part 1 of 2)

2. Many of the images in this powerpoint presentation are from Bioinformatics and Functional Genomics by Jonathan Pevsner (ISBN 0-471-21004-8). Copyright © 2003 by John Wiley & Sons, Inc.John Wiley & Sons, Inc These images and materials may not be used without permission from the publisher. We welcome instructors to use these powerpoints for educational purposes, but please acknowledge the source. The book has a homepage at http://www.bioinfbook.orghttp://www.bioinfbook.org Including hyperlinks to the book chapters. Copyright notice

3. Outline for today Protein analysis and proteomics Individual proteins Protein families Physical properties Localization Function Large-scale protein analysis 2D protein gels Yeast two-hybrid Rosetta Stone approach Pathways

4. protein Page 224 RNA DNA

5. protein [1] Protein families Page 224

6. protein [1] Protein families [2] Physical properties Page 224

7. protein [1] Protein families [2] Physical properties Page 224 [3] Protein localization

8. protein [1] Protein families [4] Protein function [2] Physical properties Page 224 [3] Protein localization

9. protein [1] Protein families [4] Protein function [2] Physical properties Page 224 [3] Protein localization Gene ontology (GO): --cellular component --biological process --molecular function

10. Perspective 1: Protein domains and motifs Page 225

11. Definitions Signature: a protein category such as a domain or motif Page 225

12. Definitions Signature: a protein category such as a domain or motif Domain: a region of a protein that can adopt a 3D structure a fold a family is a group of proteins that share a domain examples: zinc finger domain immunoglobulin domain Motif (or fingerprint): a short, conserved region of a protein typically 10 to 20 contiguous amino acid residues Page 225

13. 15 most common domains (human) Zn finger, C2H2 type1093 proteins Immunoglobulin1032 EGF-like471 Zn-finger, RING458 Homeobox417 Pleckstrin-like405 RNA-binding region RNP-1400 SH3394 Calcium-binding EF-hand392 Fibronectin, type III300 PDZ/DHR/GLGF280 Small GTP-binding protein 261 BTB/POZ236 bHLH226 Cadherin226 Page 227

14. 15 most common domains (various species) The European Bioinformatics Institute (EBI) offers many key proteomics resources: http://www.ebi.ac.uk/proteome/ Page 227

16. Definition of a domain According to InterPro at EBI ( http://www.ebi.ac.uk/interpro /): A domain is an independent structural unit, found alone or in conjunction with other domains or repeats. Domains are evolutionarily related. According to SMART (http://smart.embl-heidelberg.de): A domain is a conserved structural entity with distinctive secondary structure content and a hydrophobic core. Homologous domains with common functions usually show sequence similarities. Page 226

17. Varieties of protein domains Page 228 Extending along the length of a protein Occupying a subset of a protein sequence Occurring one or more times

18. Example of a protein with domains: Methyl CpG binding protein 2 (MeCP2) MBD Page 227 TRD The protein includes a methylated DNA binding domain (MBD) and a transcriptional repression domain (TRD). MeCP2 is a transcriptional repressor. Mutations in the gene encoding MeCP2 cause Rett Syndrome, a neurological disorder affecting girls primarily.

19. Page 228 Result of an MeCP2 blastp search: A methyl-binding domain shared by several proteins

20. Page 228 Are proteins that share only a domain homologous?

21. Example of a multidomain protein: HIV-1 pol 1003 amino acids long cleaved into three proteins with distinct activities: -- aspartyl protease -- reverse transcriptase -- integrase We will explore HIV-1 pol and other proteins at the Expert Protein Analysis System (ExPASy) server. Visit www.expasy.org/ Page 229

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24. SwissProt entry for HIV-1 pol links to many databases

25. Page 231 ProDom entry for HIV-1 pol shows many related proteins

26. Page 231 Proteins can have both domains and patterns (motifs) Domain (aspartyl protease) Domain (reverse transcriptase) Pattern (several residues) Pattern (several residues)

27. Page 232

28. Definition of a motif A motif (or fingerprint) is a short, conserved region of a protein. Its size is often 10 to 20 amino acids. Simple motifs include transmembrane domains and phosphorylation sites. These do not imply homology when found in a group of proteins. PROSITE (www.expasy.org/prosite) is a dictionary of motifs (there are currently 1600 entries). In PROSITE, a pattern is a qualitative motif description (a protein either matches a pattern, or not). In contrast, a profile is a quantitative motif description. We will encounter profiles in Pfam, ProDom, SMART, and other databases. Page 231-233

29. Perspective 2: Physical properties of proteins Page 233

30. Page 234

31. Physical properties of proteins Many websites are available for the analysis of individual proteins. ExPASy and ISREC are two excellent resources. The accuracy of these programs is variable. Predictions based on primary amino acid sequence (such as molecular weight prediction) are likely to be more trustworthy. For many other properties (such as posttranslational modification of proteins by specific sugars), experimental evidence may be required rather than prediction algorithms. Page 236

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39. Syntaxin, SNAP-25 and VAMP are three proteins that interact via coiled-coil domains

40. Introduction to Perspectives 3 and 4: Gene Ontology (GO) Consortium Page 237

41. The Gene Ontology Consortium An ontology is a description of concepts. The GO Consortium compiles a dynamic, controlled vocabulary of terms related to gene products. There are three organizing principles: Molecular function Biological process Cellular compartment You can visit GO at http://www.geneontology.org. There is no centralized GO database. Instead, curators of organism-specific databases assign GO terms to gene products for each organism. Page 237

42. Page 241 GO terms are assigned to LocusLink entries

43. Page 241

46. The Gene Ontology Consortium: Evidence Codes ICInferred by curator IDAInferred from direct assay IEAInferred from electronic annotation IEPInferred from expression pattern IGIInferred from genetic interaction IMPInferred from mutant phenotype IPIInferred from physical interaction ISSInferred from sequence or structural similarity NASNon-traceable author statement NDNo biological data TASTraceable author statement Page 240

47. Perspective 3: Protein localization Page 242

48. protein Protein localization Page 242

49. Protein localization Proteins may be localized to intracellular compartments, cytosol, the plasma membrane, or they may be secreted. Many proteins shuttle between multiple compartments. A variety of algorithms predict localization, but this is essentially a cell biological question. Page 240

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55. Localization of 2,900 yeast proteins Michael Snyder and colleagues incorporated epitope tags into thousands of S. cerevisiae cDNAs, and systematically localized proteins (Kumar et al., 2002). See http://ygac.med.yale.edu for a database including 2,900 fluorescence micrographs. Page 243

56. Perspective 4: Protein function Page 243

57. Protein function Function refers to the role of a protein in the cell. We can consider protein function from a variety of perspectives. Page 243

58. 1. Biochemical function (molecular function) RBP binds retinol, could be a carrier Page 245

59. 2. Functional assignment based on homology RBP could be a carrier too Other carrier proteins Page 245

60. 3. Function based on structure RBP forms a calyx Page 245

61. 4. Function based on ligand binding specificity RBP binds vitamin A Page 245

62. 5. Function based on cellular process DNARNA RBP is abundant, soluble, secreted Page 245

63. 6. Function based on biological process RBP is essential for vision Page 245

64. 7. Function based on “proteomics” or high throughput “functional genomics” High throughput analyses show... RBP levels elevated in renal failure RBP levels decreased in liver disease Page 245

65. Functional assignment of enzymes: the EC (Enzyme Commission) system Oxidoreductases1,003 Transferases1,076 Hydrolases1,125 Lyases356 Isomerases156 Ligases126 Page 246

66. Functional assignment of proteins: Clusters of Orthologous Groups (COGs) Information storage and processing Cellular processes Metabolism Poorly characterized Page 247

67. Functional assignment of proteins: Clusters of Orthologous Groups (COGs) Information storage and processing Cellular processes Metabolism Poorly characterized (Most useful for prokaryotes) Page 247

68. This lecture continues in part 2 with a discussion of two dimensional gels and the yeast two-hybrid system