Year of Award

2015

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Biochemistry & Biophysics

Department or School/College

Biochemistry and Biophysics Program

Committee Chair

Bruce E. Bowler

Commitee Members

J. Stephen Lodmell, Edward Rosenberg, J. B. Alexander (Sandy) Ross, Stephen R. Sprang

Keywords

alkaline conformational transition, apoptosis, conformationally gated electron transfer, Cytochrome c, X-ray crystallography

Abstract

The field of protein biochemistry has been dominated by the dogma that a protein sequence yields a 3-dimensional structure important for a singular function. More modern insights are beginning to demonstrate that proteins are not static structures. Rather, proteins undergo numerous conformational fluctuations yielding an ensemble of conformational populations. Conformational change can result in changed or altered protein function. Small or large energetic barriers existing between conformers regulate the ease with which a protein can sample alternative conformations. In the dissertation work presented here, alternative conformations of yeast iso-1-cytochrome c are investigated with particular emphasis on heme crevice loop dynamics. The heme crevice loop, or O-loop D, is a highly conserved, dynamic region. Conformational changes in O-loop D lead to altered electron transfer and peroxidase activity in cytochrome c (Cytc). As Cytc participates in both the electron transport chain and functions as a peroxidase during apoptosis, it is important to understand how this conformational change is regulated. Within O-loop D we investigate the effects of a trimethyllysine to alanine mutation and a destabilizing leucine to alanine mutation at residues 72 and 85, respectively, on heme crevice dynamics. Residue 72 plays an important role in regulating access to alternative heme crevice conformers. Of particular interest, residue 72 plays a role in regulating access to a peroxidase capable conformer of Cytc, a function of Cytc during the early stages of apoptosis. We have also solved the structure of the first monomeric Cytc structure in a peroxidase capable conformer, as well as, a dimeric Cytc structure with CYMAL-6 protruding into the interior of the heme cavity, in a manner potentially similar to the Cytc/cardiolipin interaction.

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© Copyright 2015 Levi James McClelland