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Proteomics

Proteomics. Presenting: Yaniv Loewenstein. Say What?. Proteome - the entire complement of proteins produced in a cell or organism. Proteomics – an emerging scientific discipline encompassing any method for the large scale analysis of proteins. Why proteomics?.

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Proteomics

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  1. Proteomics Presenting: Yaniv Loewenstein

  2. Say What? • Proteome - the entire complement of proteins produced in a cell or organism. • Proteomics – an emerging scientific discipline encompassing any method for the large scale analysis of proteins.

  3. Why proteomics? • Proteome analysis is conceptually attractive because ofits potential to determine properties of biological systems thatare not apparent by DNA or mRNA sequence analysis alone. • quantity of protein expression. • the subcellularlocation. • the state of modification. • the association withligands. • the rate of change with time of such properties.

  4. Genome vs. Proteome Complexity The proteome is much more complex than the genome. • Alternative Splicing. • RNA editing. • Alternative use of different promoters and terminators. • Post Translational Modifications. 1 Gene  various proteins

  5. Genome vs. Proteome Complexity – (continued) • Several genomes were solved but no eukaryotic proteomes yet. • Current efforts: + PPAP - the plant proteome annotation project. (Arabidopsis thaliana) + D.melanogaster , M. musculus, S.cerevisiae and more. • Deinococcus radiodurans - proteome was complete recently.

  6. Major Tools • 2D Gels. • Pulse-Chase experiments. • Chromatography. • Mass Spectrometry. • Immunoprecipitation. • Bioinformatics and databases YPD, CATH, SCOPE, SWISSPROT, BLAST…

  7. 2d gel

  8. Pulse Chase Q:What is meant by a pulse-chase experiment? A: Pulse:A brief exposure to some labeling condition (i.e. radioactive labeling). Chase:Removal of the label and observation of the labeled material. => follow the fate of the labeled material.

  9. Today’s Topics • Identification of in vivo substrates of the chaperonin GroEL Walid A. Houry , Dmitirij Frishman, Christoph Eckerskorn, Friedrich Lottspeich & F. Ulrich Hartl. • A sampling of the yeast proteome.Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels JI. • Correlation between protein and mRNA abundance in yeast.Gygi SP, Rochon Y, Franza BR, Aebersold R

  10. Protein Folding 3D fold of a protein is determined by the amino-acid sequence. • Some proteins can reach their folded states spontaneously. • The efficiency of folding is often limited by the side reaction of aggregation. • Misfolding and aggregation of proteins is prevented by molecular chaperones.

  11. Hsp70 *Heat shock protection. *escort translocation of unfolded proteins. *Mr ~ 70 kDa Chaperonins * Elaborate protein complexes. * Essential for correct folding of some newly synthesized proteins. Molecular chaperones - what? Definition: Proteins that interact with partially folded or improperly folded polypepetides, facilitating correct folding pathways or providing microenvironments in which folding can occur.

  12. GroEL - I • Essential. In E. Coli. • Structure: • homo-oligomer. • 2 * heptamer rings. • cofactor GroES + ATP. • some proteins may require several rounds. • Hydrophobic interactions.

  13. GroEl cycle Mechanism.

  14. Research Goals • Define a Group of GroEL Subtrates. • Search for common motifs in the GroEL substrate proteins.

  15. Experiment synopsis • Pulse-chase labeling. • At different times of chase, cells were lysed in the presence of EDTA. • GroEL–substrate complexes were isolated by immunoprecipitation with anti-GroEL antibodies. • ATP + GroES added in vitro. • Total soluble cytoplasmic proteins and GroEL-bound proteins were separated on 2D gels. • Control experiments.

  16. Substrates • 250–300 proteins out of a total of 2,500 were observed in complex with GroEL immediately upon labeling. • About half of these proteins were still detectable on GroEL after 10 min of chase , though in much lower amounts relative to GroEL. • Proteins that interact only very transiently with GroEL may have escaped detection in this analysis.

  17. A defined set of GroEL substrates – Gel

  18. GroEL substrates - pI and Mr The pI distributions of total soluble cytoplasmic proteins and of GroEL substrates were found to be very similar. E(total Mr) = 37kD E(GroEL sub Mr) = 45kD BUT ~20kD<Mr<~60kD

  19. GroEL substrates - Kinetics a. 160 proteins (Mr < 60k) released completely with time constants between 20s and 2 min. b. 100 proteins (Mr < 60k), a fraction of the substrates remained associated. c. Several larger proteins (Mr > 60k) were inefficiently released from the chaperonin.

  20. A defined set of GroEL substrates – Gel

  21. Additional experiments • Proteins return to GroEL for additional cycles. • Heat stress conditions.

  22. GroEL substrates – Identification. • Analyze spots - MS. • 52 of the most abundant, were unequivocally IDed. * RNA poly subunits * E.F Tu * tRNA synthetases in accord with previous studies.

  23. GroEl Substrates – Common Motif. • Run PSI-BLAST for consensus sequence or clusters of thereof => 0, null, gurnicht mit gurnicht!  • domains were classified independently through sequence homology to domains in SCOP & CATH. => 18-24 proteins.  and the plot thickens ….

  24. A preferred structural Motif • GroEL substrates preferentially contain several αβ domains compared with E. coli proteins. • Of the multidomain GroEL substrates, 13 of 17 have at least two αβ domains. • No significant preference for other 3D structures was found. • Several model substrates of GroEL, typically used for in vitro studies, belong to this category of αβ proteins.

  25. Graph

  26. Substrate interaction with GroEL • Preferred domain topology, provides insight into why and how these proteins interact with the chaperonin. • Proteins with two or more αβ domains, may be particularly prone to aggregation. • Hydrophobic interactions propose possible mechanism.

  27. Possible Motivation • Biotechnolgy Improve the folding efficiency of foreign proteins expressed in E. coli. • Better Understanding of protein Folding Mechanisms in eukaryotes.

  28. Coffee

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