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Protein folding in the cell: The Hsp90 Chaperone Machine Stefan Rüdiger Utrecht, 26 February 2009

Master Biomolecular Sciences 2008/2009 Master course “Membrane Biogenesis, Protein Folding and Sorting”. Protein folding in the cell: The Hsp90 Chaperone Machine Stefan Rüdiger Utrecht, 26 February 2009. R099. Protein folding and unfolding. Protein unfolding.

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Protein folding in the cell: The Hsp90 Chaperone Machine Stefan Rüdiger Utrecht, 26 February 2009

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  1. Master Biomolecular Sciences 2008/2009 Master course “Membrane Biogenesis, Protein Folding and Sorting” Protein folding in the cell: The Hsp90 Chaperone MachineStefan RüdigerUtrecht, 26 February 2009 R099

  2. Protein folding and unfolding

  3. Protein unfolding

  4. Un-boiling egg white with Guanidinium Chloride

  5. Unscrambling egg white with Guanidinium Chloride

  6. Unscrambling egg white with Guanidinium Chloride

  7. Unscrambling egg white with Guanidinium Chloride Buffer 7 M GdmCl 7 M GdmCl heat Hermsen et al (2006)

  8. 2. Protein folding in the cell

  9. DNA The Central Dogma (Crick 1958) RNA Protein

  10. Biochemical Pathways

  11. 3. Molecular Chaperones

  12. Processes involving chaperones degradation folding aggregation translocation

  13. Many heat shock proteins are chaperones Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp Specialised chaperones

  14. Many heat shock proteins are chaperones Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp Specialised chaperones

  15. Many heat shock proteins are chaperones cytosol 4 humans Hsp90 Hsp90 Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp Specialised chaperones ER Grp94 mitochondria Trap1 1 E. coli HtpG

  16. 4. Hsp90 - knowns

  17. N N M M C C Hsp90 • 83 KDa • 166 KDa dimer • ATPase in N-terminal domain • Substrate binding site unknown

  18. ~20000 genes ~20000 proteins The Hsp90 substrate pool Hsp90 ?

  19. ~20000 genes ~20000 proteins The Hsp90 substrate pool ~130 Hsp90 substrates Hsp90 ?

  20. ~20000 genes ~20000 proteins The Hsp90 substrate pool ~130 Hsp90 substrates ~60 kinases Transcription factors Hsp90 ? Steroid receptors p53 CFTR

  21. 5. Hsp90 - unknowns

  22. ~20000 genes ~20000 proteins 1. Ratio mismatch ~130 Hsp90 substrates ~60 kinases Transcription factors Hsp90 ? Steroid receptors p53 CFTR

  23. ~20000 genes ~20000 proteins 2. Substrate diversity ~130 Hsp90 substrates ~60 kinases Transcription factors Hsp90 ? Steroid receptors p53 CFTR

  24. ~20000 genes ~20000 proteins 3. What is up with kinases? ~130 Hsp90 substrates ~60 kinases Transcription factors Hsp90 ? Steroid receptors p53 CFTR

  25. 6. Hsp90 structures

  26. Hsp90-N (human) Stebbins, Russo, Schneider, Rosen, Hartl & Pavletich, Cell 89:239-250 (1997)

  27. Hsp82 (yeast Hsp90; AMP-PNP, p23) Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:1013-1017

  28. HtpG (E. coli Hsp90, ATP-free) Shiau, Harris, Southworth & Agard, Cell 127:329-340 (2006)

  29. The Hsp90 substrate cycle Richter & Buchner Cell 127:251-253 (2006)

  30. Hsp90 conformations Dollins, Warren, Immormino & Gewirth Mol Cell 28:41-56 (2007)0

  31. SAXS indicates Hsp90 flexibility Krukenberg, Förster, Rice, Sali & Agard Structure 16:755-765 (2008)

  32. 7. Hsp90 interaction with substrates

  33. 110 115 120 125 130 Hsp90 can be monitored by NMR 15N CRINEPT-TROSY 158 µM Hsp90 Dimer (15N,2H) 1H 10 9 8 7

  34. 15N 110 115 120 125 130 1H 10 9 8 7 HSQC spectrum of free p53 core domain p53core (free) HSQC 100 µM p53core (15N,2H)

  35. 15N 110 115 120 125 130 1H 10 9 8 7 p53core bound to Hsp90 is unfolded p53core (Hsp90) CRINEPT-TROSY 100 µM p53core (15N,2H) 100 µM p53core (15N,2H) 147 µM Hsp90 Dimer (2H)

  36. 15N 110 115 120 125 130 1H 10 9 8 7 Signals of urea-unfolded p53 cluster in same region p53core (Hsp90) CRINEPT-TROSY p53core (urea) HSQC 100 µM p53core (15N,2H) 100 µM p53core (15N,2H) 147 µM Hsp90 Dimer (2H)

  37. p53core bound to Hsp90 …is unfolded

  38. Kinase targeting: Hsp90, cdc37 (targeting factor) and Cdk4 (substrate) 4 humans 1 E. coli Vaughan, Gohlke, Sobott, Good, Ali, Prodromou, Robinson, Saibil & Pearl Mol Cell 23:697-707 (2006)

  39. 8. The Hsp90 cycle

  40. The Hsp90 substrate cycle Richter & Buchner Cell 127:251-253 (2006)

  41. 9. Hsp90 co-factors

  42. Substrate targeting factor Cdc37 Roe, Ali, Meyer, Vaughan, Panaretou, Piper, Prodromou & Pearl Cell 127:329-340 (2006)

  43. Kinase targeting: Hsp90, cdc37 (targeting factor) and Cdk4 (substrate) 4 humans 1 E. coli Vaughan, Gohlke, Sobott, Good, Ali, Prodromou, Robinson, Saibil & Pearl Mol Cell 23:697-707 (2006)

  44. ATP hydrolysis: Hsp90 and Aha1 Meyer, Prodromou, Liao, Roe, Vaughan, Vlasic, Panaretou, Piper, & Pearl EMBO J 23:1402-1410 (2004)

  45. Nucleotide exchange: Hsp90 and p23 4 humans 1 E. coli Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:1013-1017

  46. Coupling to degradation: Hsp90 and CHIP Zhang, Windheim, Roe, Peggie, Cohen, Prodromou & Pearl Mol Cell 20:525-538 ()2005)

  47. The Hsp90 substrate cycle Richter & Buchner Cell 127:251-253 (2006)

  48. Hsp90-dependend refolding of citrate synthase Jakob, Lilie, Meyer, Buchner JBC 270:7288-7294 (1995)

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