Complex Analysis - George Cain - Georgia Institute of Technology
Jaya George Department of Chemistry The University of Georgia
-
Upload
georgina-vernon -
Category
Documents
-
view
14 -
download
0
description
Transcript of Jaya George Department of Chemistry The University of Georgia
![Page 1: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/1.jpg)
Determination of Determination of Chaperone Activity Chaperone Activity
through through in vivoin vivo testing of testing of GroEL/GroES, DnaK/DnaJ, GroEL/GroES, DnaK/DnaJ,
and HscA/HscB and HscA/HscB suppression of missense suppression of missense
mutationsmutationsJaya GeorgeJaya George
Department of ChemistryDepartment of Chemistry
The University of GeorgiaThe University of Georgia
![Page 2: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/2.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 3: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/3.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 4: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/4.jpg)
BackgroundBackground
• Self-assembly vs. Assisted-assemblySelf-assembly vs. Assisted-assembly
• AnfinsenAnfinsen
![Page 5: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/5.jpg)
Illustration of Protein FoldingIllustration of Protein Folding
![Page 6: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/6.jpg)
Background continuedBackground continued
• Self-assembly vs. Assisted-Self-assembly vs. Assisted-assemblyassembly
• AnfinsenAnfinsen
• LaskeyLaskey
• EllisEllis
![Page 7: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/7.jpg)
Background continuedBackground continued
• Definition of chaperonesDefinition of chaperones– Chaperones are assigned as a family of proteins that Chaperones are assigned as a family of proteins that
assist other proteins to fold into their active forms.assist other proteins to fold into their active forms.– If chaperones in fact exist, their functions would include If chaperones in fact exist, their functions would include
prevention of inactive structural forms as well as aiding prevention of inactive structural forms as well as aiding in the reversal of misfolding that result from stresses.in the reversal of misfolding that result from stresses.
![Page 8: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/8.jpg)
Background continuedBackground continued
• Self-assembly vs. Assisted-assemblySelf-assembly vs. Assisted-assembly• AnfinsenAnfinsen• LaskeyLaskey• EllisEllis
• Results from previous Results from previous studiesstudies
![Page 9: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/9.jpg)
Previous Previous In VitroIn Vitro Testing TestingProtein Tested Protein Tested GroEL GroES DnaKJ Ref GroEL GroES DnaKJ Ref
• b-galactosidaseb-galactosidase ++ NTNT NTNT (2)(2)• Human carbonic anhydrase IIHuman carbonic anhydrase II ++ ++ NTNT (18)(18)• Human pro-urokinaseHuman pro-urokinase ++ NTNT NTNT (19)(19)• Firefly LuciferaseFirefly Luciferase NTNT NTNT ++ (15)(15)• CatalaseCatalase ++ NTNT NTNT (13)(13)• Glycerol dehydrogenaseGlycerol dehydrogenase ++ NTNT NTNT (14)(14)• Mitochondrial rhodanaseMitochondrial rhodanase ++ NTNT NTNT (16)(16)• Ornithine transcarbamylaseOrnithine transcarbamylase ++ ++ NTNT (20)(20)• Glucose-6-phosphate Glucose-6-phosphate dehydrogenasedehydrogenase ++ NTNT NTNT (12)(12)• Glutamine synthetaseGlutamine synthetase ++ ++ NTNT (9)(9)• Lambda repressorLambda repressor NTNT NTNT ++• TryptophanaseTryptophanase ++ -- NTNT (17)(17)
![Page 10: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/10.jpg)
Previous Previous In VivoIn Vivo Studies Studies
Protein Tested GroEL GroES DnaKJRef
• S1 Dihydrofolate reductase + + NT (6)• Tyrosine kinase + + + (4)• Ribulose-biphosphate carboxylase NT NT + (5)• Human growth hormone - + + (3)• E. coli glutamate racemase + + NT (1)
![Page 11: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/11.jpg)
Assisted AssemblyAssisted Assembly
![Page 12: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/12.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 13: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/13.jpg)
GoalsGoals
• Establish method of testing Establish method of testing chaperones chaperones in vivoin vivo..
• Observe trends in chaperone effect.Observe trends in chaperone effect.• Characterize unstudied chaperone Characterize unstudied chaperone
HscAB.HscAB.• Make comparisons between Make comparisons between
GroEL/GroES, DnaK/DnaJ, and GroEL/GroES, DnaK/DnaJ, and HscA/HscABHscA/HscAB
![Page 14: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/14.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 15: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/15.jpg)
MethodsMethods• Pull freezer strains of missense Pull freezer strains of missense
mutations.mutations.• Isolate pure culturesIsolate pure cultures• Generate Competent cellsGenerate Competent cells• Transform cells with target plasmidTransform cells with target plasmid• Patch cells and check for restored Patch cells and check for restored
activity.activity.
![Page 16: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/16.jpg)
Sample PatchSample Patch
![Page 17: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/17.jpg)
Methods ContinuedMethods Continued
• Check for overexpression through Check for overexpression through protein gels.protein gels.
• Patch cells and check for Patch cells and check for restored activity.restored activity.
![Page 18: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/18.jpg)
Protein GelProtein Gel
![Page 19: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/19.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 20: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/20.jpg)
HisD Mutant Suppressionwith GroELS
Suppressionwith DnaKJ
Suppressionwith HscAB
HisD64 + ++ -HisD68 ++ - -HisD74 ++ - -HisD88 ++ - -HisD111 ++ - -HisD223 + + -HisD226 ++ - -HisD237 + - -HisD248 + + -HisD274 ++ - -HisD295 ++ - -HisD412 ++ - -HisD450 ++ - -
Results for His D missense mutationsResults for His D missense mutations
![Page 21: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/21.jpg)
ResultsResults for LacZ missense mutationsfor LacZ missense mutations
LacZ Mutant Suppressionwith GroELS
Suppressionwith DnaKJ
Suppressionwith HscAB
LacZ172 - - +
LacZ173 - + ++
LacZ174 - + ++
LacZ190 - - +
LacZ202 - ++ ++
LacZ211 - ++ ++
LacZ220 - - +
LacZ225 - - ++
![Page 22: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/22.jpg)
OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion
![Page 23: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/23.jpg)
DiscussionDiscussion• The first mutants analyzed showed The first mutants analyzed showed
chaperone overexpression, but not chaperone overexpression, but not at the levels desired.at the levels desired.
• DNA from 100 mutant strains have DNA from 100 mutant strains have again been isolated and purified.again been isolated and purified.
• The next step in this research The next step in this research would be to transform the would be to transform the reconstructed chaperone plasmid reconstructed chaperone plasmid into the mutant strains. into the mutant strains.
![Page 24: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/24.jpg)
Discussion ContinuedDiscussion Continued• Preliminary results show that the Preliminary results show that the in in
vivovivo method of testing is feasible method of testing is feasible and practical.and practical.
• The resulting data will allow The resulting data will allow concrete trends in chaperone concrete trends in chaperone activity to be established.activity to be established.
![Page 25: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/25.jpg)
Discussion continuedDiscussion continued• Efforts towards characterization of Efforts towards characterization of
the unstudied HscAB complex will the unstudied HscAB complex will continue.continue.
• Preliminary results indicate that it Preliminary results indicate that it may be possible to interchange may be possible to interchange chaperones as a means of chaperones as a means of reversing the same mutations.reversing the same mutations.
![Page 26: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/26.jpg)
SummarySummary• Chaperones have been identified and Chaperones have been identified and
proven to aid in protein folding.proven to aid in protein folding.• In vivo In vivo studies are currently the studies are currently the
means by which to obtain the most means by which to obtain the most accurate trends in chaperone accurate trends in chaperone activity.activity.
• Eventual applications of this research Eventual applications of this research may include reversal of missense may include reversal of missense mutations that cause disorders such mutations that cause disorders such as Sickle Cell Anemia.as Sickle Cell Anemia.
![Page 27: Jaya George Department of Chemistry The University of Georgia](https://reader035.fdocuments.in/reader035/viewer/2022062517/56813354550346895d9a6541/html5/thumbnails/27.jpg)
Special Thanks to Dr. Elliot Special Thanks to Dr. Elliot Altman and Ryan Schwaner Altman and Ryan Schwaner
for their leadership and for their leadership and guidance during this project. guidance during this project.