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

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

BackgroundBackground

• Self-assembly vs. Assisted-assemblySelf-assembly vs. Assisted-assembly

• AnfinsenAnfinsen

Illustration of Protein FoldingIllustration of Protein Folding

Background continuedBackground continued

• Self-assembly vs. Assisted-Self-assembly vs. Assisted-assemblyassembly

• AnfinsenAnfinsen

• LaskeyLaskey

• EllisEllis

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.

Background continuedBackground continued

• Self-assembly vs. Assisted-assemblySelf-assembly vs. Assisted-assembly• AnfinsenAnfinsen• LaskeyLaskey• EllisEllis

• Results from previous Results from previous studiesstudies

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)

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)

Assisted AssemblyAssisted Assembly

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

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

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

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.

Sample PatchSample Patch

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.

Protein GelProtein Gel

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

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

ResultsResults for LacZ missense mutationsfor LacZ missense mutations

LacZ Mutant Suppressionwith GroELS

Suppressionwith DnaKJ

Suppressionwith HscAB

LacZ172 - - +

LacZ173 - + ++

LacZ174 - + ++

LacZ190 - - +

LacZ202 - ++ ++

LacZ211 - ++ ++

LacZ220 - - +

LacZ225 - - ++

OverviewOverview• BackgroundBackground• GoalsGoals• MethodsMethods• ResultsResults• DiscussionDiscussion

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.

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.

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.

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.

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.