An Empirical Test of Evolution in Rhodopsins by Examination of Novel Folds

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dc.contributor.advisor Theobald, Douglas
dc.contributor.author Mackin, Kristine
dc.date.accessioned 2014-05-21T20:18:01Z
dc.date.available 2014-05-21T20:18:01Z
dc.date.issued 2014
dc.identifier.uri http://hdl.handle.net/10192/27237
dc.description.abstract Type I and type II rhodopsins share an identical fold, but have no detectable sequence similarity. The evolutionary relationship between these proteins has been debated for decades. Convergent evolution predicts that this shared fold originated due to functional restraints. Homology is typically established when proteins have sequence similarity, although a lack of similarity is a prediction of descent from an ancient common ancestor. Most recent reviews claim these proteins originated through convergent evolution, but current data is not sufficient to definitively conclude that these proteins are not homologous. In this work, I test the key prediction of convergent evolution. That is, that only one fold is capable of targeting to the membrane, folding correctly, binding retinal, and undergoing the light-activated proton pumping to provide a critical energy source for the archaeon. I designed and characterized seven novel fold mutants and one point mutant that changes the position of the strictly conserved lysine required for covalent binding to the retinal cofactor. These proteins all target to the cell membrane, fold, and bind retinal, causing a color change upon expression in the E. coli cells. The seven fold mutants all absorb within 2 nm of the wild-type, indicating that the protein is forming the correct helical contacts and binding retinal. When reconstituted into liposomes, the mutants all exhibit light-activated proton pumping activity from 40-170% of the wild-type. Only one of the lysine point mutants, A53K/K216A, has reproducible proton pumping activity at about 11% of the wild-type. The ability of these seven novel folds and the single point mutant indicated that evolution is not constrained to one particular fold, so convergence is unlikely. Therefore I propose that type I and type II rhodopsins share an ancient common ancestor and are homologous.
dc.description.sponsorship Brandeis University, Graduate School of Arts and Sciences
dc.format.mimetype application/pdf
dc.language English
dc.language.iso eng
dc.publisher Brandeis University
dc.relation.ispartofseries Brandeis University Theses and Dissertations
dc.rights Copyright by Kristine Ann Mackin 2014
dc.subject Evolution
dc.subject rhodopsin
dc.subject GPCR
dc.subject bacteriorhodopsin
dc.subject membrane protein
dc.title An Empirical Test of Evolution in Rhodopsins by Examination of Novel Folds
dc.type Thesis
dc.contributor.department Department of Biochemistry
dc.degree.name PhD
dc.degree.level Doctoral
dc.degree.discipline Biochemistry
dc.degree.grantor Brandeis University, Graduate School of Arts and Sciences


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