Evaluation of protein aggregation and organismal fitness
| dc.contributor.advisor | Ellington, Andrew D. | en |
| dc.contributor.committeeMember | Marcotte, Edward | en |
| dc.contributor.committeeMember | Whiteley, Marvin | en |
| dc.contributor.committeeMember | Wilke, Claus O. | en |
| dc.contributor.committeeMember | Willets, Katherine | en |
| dc.creator | Stovall, Gwendolyn Motz | en |
| dc.date.accessioned | 2011-06-01T21:13:08Z | en |
| dc.date.available | 2011-06-01T21:13:08Z | en |
| dc.date.available | 2011-06-01T21:13:24Z | en |
| dc.date.issued | 2011-05 | en |
| dc.date.submitted | May 2011 | en |
| dc.date.updated | 2011-06-01T21:13:24Z | en |
| dc.description | text | en |
| dc.description.abstract | In quiescent yeast, the widespread reorganization of cytosolic proteins into punctate has been observed (Narayanaswamy et al. 2009). We seek to better understand and describe this reorganization, which we hypothesize to be a protein aggregation phenomenon. To test this hypothesis, we examined mutant proteins (Ade4p protein variants) in yeast with predicted non-native aggregation propensities and measured their punctate formation kinetics. Monitoring punctate formation kinetics involved the validation of an automated quantification technique using an Amnis ImageStream imaging flow cytometer. The automated punctate counts were strongly correlated with the manual punctate counts, with usual R² values of 0.99 or better, but evaluated 50-fold more cells per run. Fitness evaluations of the mutant yeast in the form of growth curves and batch competition experiments revealed the slowed growth of the Ade4-1286 strain and the functional inequality to the wild type strain of the Ade4-mtoin2034, Ade4-mtoin2105, and Ade4-2800 strains in competition experiments, especially when the mutants were forced to generate their own adenine. Subsequent structural analysis of the mutant proteins revealed destabilizing mutations for 4 of the 6 mutant proteins with 2 of the mutations classified as significantly destabilizing ([delta][delta]G >2 kcal/mol). We concluded that the reduction in protein fitness was likely due to the destabilizing effects of the mutations. Evaluation of the punctate formation kinetics revealed little difference between strains in the rate of punctate formation. Further examination revealed the wild type Ade4p and all of the mutants (with the exception of the Ade4-1286 mutant) were predicted to have similar aggregation propensities according to a secondary aggregation predicting algorithm (Zyggregator, Pawar et al. 2005). Additionally, solvent accessibility calculations estimate ~3-19% of the side chain surface area to be solvent accessible, which indicates proximity of mutations to the protein surface. However, mutating buried amino acids likely would have generated a greater disturbance (Matthews 1993, Tokuriki et al. 2007). We concluded that the mutations, although destabilizing, altered the aggregation propensity very little. Deletion of chaperone proteins (Hsp82p, Hsc82p, and Ssa1p) revealed no difference in the Ade4-GFP punctate formation kinetics, although a slight kinetic difference was detected in the chaperone (Hsp82p) knockout, Gln1-GFP strain and the wild type strain. While further workup is necessary in the chaperone knockout, Gln1-GFP work, the initial results are promising and suggest the involvement of protein folding machinery in punctate formation. | en |
| dc.description.department | Biochemistry | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.uri | http://hdl.handle.net/2152/ETD-UT-2011-05-2841 | en |
| dc.language.iso | eng | en |
| dc.subject | Protein aggregation | en |
| dc.subject | Phosphoribosylpyrophosphate amidotransferase | en |
| dc.subject | Ade4 | en |
| dc.subject | TANGO algorithm | en |
| dc.subject | Saccharomyces | en |
| dc.subject | Punctate | en |
| dc.title | Evaluation of protein aggregation and organismal fitness | en |
| dc.type.genre | thesis | en |
| thesis.degree.department | Biochemistry | en |
| thesis.degree.discipline | Biochemistry | en |
| thesis.degree.grantor | University of Texas at Austin | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |