Gatekeeper Residues Control Peroxidase Activity of Cytochrome c in Apoptosis

有了这个奖项，化学部的生命过程化学项目资助蒙大拿大学的布鲁斯·鲍勒博士测试一个基于结构的假设，该假设是关于控制细胞凋亡早期阶段所需的细胞色素C过氧化物酶活性的因素。 特别是，该项目将测试酵母和人细胞色素C序列的72，81和83位残基的空间体积在调节凋亡过程中过氧化物酶活性所需的蛋白质的替代构象的可及性中的作用。将使用pH跳跃和构象门控电子转移动力学方法、室温X射线晶体学、过氧化物酶活性测定和心磷脂纳米盘的结合亲和力评估这些位置处的突变对该替代构象异构体群体的影响。还将使用构象门控电子转移方法确定构象异构体在心磷脂纳米盘上的分布。结构-功能范式，它表明蛋白质具有为单一功能而设计的独特结构，主导着生物化学教科书。 最近的研究表明，许多蛋白质可以访问一个以上的结构，使它们能够执行多种功能。 细胞色素C参与电子传递链（需氧生物体中的主要能量储存途径）和细胞凋亡（程序性细胞死亡）（高等生物体如哺乳动物的生长和发育中的关键过程）。 这项研究将提供一个分子水平的理解，如何在细胞色素C中控制这两种功能所需的两种结构之间的转换。 本科生和研究生将接受蛋白质变体制备，高分辨率X射线晶体学和测量交替细胞色素C结构之间的相对稳定性和转换速率的方法的专门培训。作为该项目的一部分，Bowler博士将与美国原住民研究实验室，斯隆研究生土著合作伙伴计划以及蒙大拿大学的Payne Family Native American Center合作，让美国原住民学生参与蒙大拿大学生物化学实验室的研究。

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.