Evolution of Cytochrome c for Optimal Apoptotic Peroxidase Activity

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Programmed cell death (apoptosis) is a critical biochemical pathway that controls the growth and development of different tissues within an organism. An important early step in apoptosis is the oxidation of the mitochondrial membrane lipid, cardiolipin, by reactive oxygen species, including peroxides. This oxidation activity, which acts as an effective apoptotic on/off switch, is facilitated by the peroxidase activity of the protein cytochrome c. The intrinsic peroxidase activity of cytochrome c must be minimized so as not to accidentally trip the switch. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Bruce E. Bowler from the University of Montana to investigate how the amino acid sequence of cytochrome c is optimized for its peroxidase function in apoptosis. This grant aims to dissect how sequence differences between yeast (no apoptotic pathway) and human (functional apoptotic pathway) cytochrome c have perfected this switch to be off when the protein is not interacting with cardiolipin and on when it interacts with cardiolipin. This project provides training for two graduate students in molecular biology methods, protein expression, biophysical methods, structural biology and single-molecule methods so that they can develop into productive scientists enabling the growth of our scientific infrastructure. The project also develops visually-exciting educational materials for a science museum in Missoula, MT that show how proteins carry out their functions, with the aim of inspiring young people to pursue careers in science.This project uses rationally-designed variants of cytochrome c to evaluate the sequence determinants that control access to the peroxidase-competent conformer of the protein. Equilibrium and stopped-flow kinetic methods are employed to probe the structural dynamics of cytochrome c variants in the presence and the absence of cardiolipin membranes using absorbance, fluorescence and circular dichroism spectroscopies, single-molecule methods and X-ray crystallography. Peroxidase activity is evaluated in the presence and absence of cardiolipin membranes to correlate variant-dependent conformational and dynamic properties characterized spectroscopically with changes in peroxidase activity.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.