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Transcript
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 Methods Results Discussion Background • Self-assembly vs. Assisted-assembly • Anfinsen Illustration of Protein Folding Background continued • Self-assembly vs. Assistedassembly Anfinsen • Laskey • Ellis • 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. Background continued • Self-assembly vs. Assistedassembly Anfinsen Laskey Ellis • Results from previous studies • • • Previous In Vitro Testing Protein Tested GroEL GroES DnaKJ Ref • • • • • • • • • • • • b-galactosidase Human carbonic anhydrase II Human pro-urokinase Firefly Luciferase Catalase Glycerol dehydrogenase Mitochondrial rhodanase Ornithine transcarbamylase Glucose-6-phosphate dehydrogenase Glutamine synthetase Lambda repressor Tryptophanase + + + NT + + + + NT + NT NT NT NT NT + NT NT NT + NT NT NT NT (2) (18) (19) (15) (13) (14) (16) (20) + + NT + NT + NT - NT NT + NT (12) (9) (17) Previous In Vivo Studies Protein Tested GroEL GroES DnaKJ Ref • S1 Dihydrofolate reductase • Tyrosine kinase • Ribulose-biphosphate carboxylase • Human growth hormone • E. coli glutamate racemase + + + + NT + (6) (4) NT + NT + + + + NT (5) (3) (1) Assisted Assembly Overview • • • • • Background Goals Methods Results Discussion 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 Overview • • • • • Background Goals Methods Results Discussion 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. Sample Patch Methods Continued • Patch cells and check for restored activity. • Check for overexpression through protein gels. Protein Gel Overview • • • • • Background Goals Methods Results Discussion Results for His D missense mutations HisD Mutant HisD64 HisD68 HisD74 HisD88 HisD111 HisD223 HisD226 HisD237 HisD248 HisD274 HisD295 HisD412 HisD450 Suppression with GroELS Suppression with DnaKJ Suppression with HscAB + ++ ++ ++ ++ + ++ + + ++ ++ ++ ++ ++ + + - - Results for LacZ missense mutations LacZ Mutant Suppression with GroELS Suppression with DnaKJ Suppression with HscAB LacZ172 - - + LacZ173 - + ++ LacZ174 - + ++ LacZ190 - - + LacZ202 - ++ ++ LacZ211 - ++ ++ LacZ220 - - + LacZ225 - - ++ Overview • • • • • Background Goals Methods Results Discussion 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. 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. 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. 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. Special Thanks to Dr. Elliot Altman and Ryan Schwaner for their leadership and guidance during this project.
