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Jaya George Department of Chemistry The University of Georgia

Determination of Chaperone Activity through in vivo testing of GroEL/GroES, DnaK/DnaJ, and HscA/HscB suppression of missense mutations. Jaya George Department of Chemistry The University of Georgia. Overview. Background Goals Methods Results Discussion. Overview. Background Goals

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Jaya George Department of Chemistry The University of Georgia

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  1. Determination of Chaperone Activity through in vivo testing of GroEL/GroES, DnaK/DnaJ, and HscA/HscB suppression of missense mutations Jaya George Department of Chemistry The University of Georgia

  2. Overview • Background • Goals • Methods • Results • Discussion

  3. Overview • Background • Goals • Methods • Results • Discussion

  4. Background • Self-assembly vs. Assisted-assembly • Anfinsen

  5. Illustration of Protein Folding

  6. Background continued • Self-assembly vs. Assisted-assembly • Anfinsen • Laskey • Ellis

  7. Background continued • Definition of chaperones • Chaperones are assigned as a family of proteins that assist other proteins to fold into their active forms. • If chaperones in fact exist, their functions would include prevention of inactive structural forms as well as aiding in the reversal of misfolding that result from stresses.

  8. Background continued • Self-assembly vs. Assisted-assembly • Anfinsen • Laskey • Ellis • Results from previous studies

  9. Previous In Vitro TestingProtein Tested GroEL GroES DnaKJ Ref • b-galactosidase + NT NT (2) • Human carbonic anhydrase II + + NT (18) • Human pro-urokinase + NT NT (19) • Firefly Luciferase NT NT + (15) • Catalase + NT NT (13) • Glycerol dehydrogenase + NT NT (14) • Mitochondrial rhodanase + NT NT (16) • Ornithine transcarbamylase + + NT (20) • Glucose-6-phosphate • dehydrogenase + NT NT (12) • Glutamine synthetase + + NT (9) • Lambda repressor NT NT + • Tryptophanase + - NT (17)

  10. Previous In Vivo Studies • Protein Tested GroEL GroES DnaKJ Ref • S1 Dihydrofolate reductase + + NT (6) • Tyrosine kinase + + + (4) • Ribulose-biphosphate • carboxylase NT NT + (5) • Human growth hormone - + + (3) • E. coli glutamate racemase + + NT (1)

  11. Assisted Assembly

  12. Overview • Background • Goals • Methods • Results • Discussion

  13. Goals • Establish method of testing chaperones in vivo. • Observe trends in chaperone effect. • Characterize unstudied chaperone HscAB. • Make comparisons between GroEL/GroES, DnaK/DnaJ, and HscA/HscAB

  14. Overview • Background • Goals • Methods • Results • Discussion

  15. Methods • Pull freezer strains of missense mutations. • Isolate pure cultures • Generate Competent cells • Transform cells with target plasmid • Patch cells and check for restored activity.

  16. Sample Patch

  17. Methods Continued • Check for overexpression through protein gels. • Patch cells and check for restored activity.

  18. Protein Gel

  19. Overview • Background • Goals • Methods • Results • Discussion

  20. Results for His D missense mutations

  21. Resultsfor LacZ missense mutations

  22. Overview • Background • Goals • Methods • Results • Discussion

  23. Discussion • The first mutants analyzed showed chaperone overexpression, but not at the levels desired. • DNA from 100 mutant strains have again been isolated and purified. • The next step in this research would be to transform the reconstructed chaperone plasmid into the mutant strains.

  24. Discussion Continued • Preliminary results show that the in vivo method of testing is feasible and practical. • The resulting data will allow concrete trends in chaperone activity to be established.

  25. Discussion continued • Efforts towards characterization of the unstudied HscAB complex will continue. • Preliminary results indicate that it may be possible to interchange chaperones as a means of reversing the same mutations.

  26. Summary • Chaperones have been identified and proven to aid in protein folding. • In vivo studies are currently the means by which to obtain the most accurate trends in chaperone activity. • Eventual applications of this research may include reversal of missense mutations that cause disorders such as Sickle Cell Anemia.

  27. Special Thanks to Dr. Elliot Altman and Ryan Schwaner for their leadership and guidance during this project.

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