Gatekeeper Residues Control Peroxidase Activity of Cytochrome c in Apoptosis

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Bruce Bowler from the University of Montana to test a structure-based hypothesis on the factors that control the peroxidase activity of cytochrome C required in the early stages of apoptosis. In particular, the project will test the role of the steric bulk of residues at positions 72, 81 and 83 of the sequence of both yeast and human cytochrome C in modulating the accessibility of an alternate conformer of the protein required for peroxidase activity during apoptosis. The effect of mutations at these positions on population of this alternate conformer will be assessed using pH jump and conformationally-gated electron transfer kinetics methods, room temperature X-ray crystallography, peroxidase activity assays and binding affinity for cardiolipin nanodiscs. The distribution of conformers on cardiolipin nanodiscs will also be determined using conformationally-gated electron transfer methods. The structure-function paradigm, which indicates that proteins have a unique structure designed for a single function, dominates biochemistry textbooks. Recent work indicates that many proteins can access more than one structure allowing them to carry out multiple functions. Cytochrome C is involved in both the electron transport chain, the primary energy storage pathway in aerobic organisms, and in apoptosis (programmed cell death) a key process in the growth and development of higher organisms such as mammals. This research will provide a molecular level understanding of how the transition between the two structures needed to carry out these two functions is controlled in cytochrome C. Undergraduate and graduate students will receive specialized training in the preparation of protein variants, in high resolution X-ray crystallography and in methods to measure the relative stability and rates of transition between alternate cytochrome C structures. As part of this project, Dr. Bowler will work with the Native American Research Laboratory, the Sloan Graduate Indigenous Partners program, and the Payne Family Native American Center at the University of Montana to involve Native American students in research in his and other biochemistry laboratories at the University of Montana.