Gatekeeper Residues Control Peroxidase Activity of Cytochrome c in Apoptosis

通过这一奖项，化学部的生命过程化学计划资助蒙大拿大学的Bruce Bowler博士测试一项基于结构的假说，该假说涉及在细胞凋亡的早期阶段所需的控制细胞色素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序列第72、81和83位的立体残基在调节细胞凋亡过程中过氧化物酶活性所需蛋白质的替代构象的可及性方面的作用。利用pH跃迁和构象门控电子转移动力学方法、室温X射线结晶学、过氧化物酶活性测定和与心磷脂纳米盘的结合亲和力来评估这些位置的突变对这种替代构象的群体的影响。心磷脂纳米盘上的构象分布也将使用构象门控电子转移方法来确定。结构-功能范式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不止一个结构，从而实现多种功能。细胞色素C既参与有氧生物中主要的能量储存途径--电子传递链，也参与了细胞凋亡(程序性细胞死亡)，后者是高等生物(如哺乳动物)生长和发育的关键过程。这项研究将在分子水平上了解细胞色素C如何控制执行这两种功能所需的两种结构之间的转变，本科生和研究生将接受有关蛋白质变体的准备、高分辨率X射线结晶学以及测量替代细胞色素C结构之间的相对稳定性和转变速率的方法的专门培训。作为该项目的一部分，鲍勒博士将与美国原住民研究实验室、斯隆研究生土著伙伴项目和蒙大拿大学佩恩家庭美国原住民中心合作，让美国原住民学生参与他和蒙大拿大学其他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.