Evolution of Cytochrome c for Optimal Apoptotic Peroxidase Activity

细胞程序性死亡（凋亡）是控制生物体内不同组织生长发育的重要生化途径。细胞凋亡的一个重要的早期步骤是线粒体膜脂（心磷脂）被活性氧（包括过氧化物）氧化。这种氧化活性，作为一个有效的凋亡开关，是由蛋白质细胞色素c的过氧化物酶活性促进的。细胞色素c固有的过氧化物酶活性必须最小化，以免意外绊倒开关。有了这个奖项，化学部门的生命过程化学项目资助了蒙大拿大学的Bruce E. Bowler博士，研究细胞色素c的氨基酸序列如何优化其过氧化物酶在细胞凋亡中的功能。这项拨款旨在分析酵母（无凋亡途径）和人类（功能性凋亡途径）细胞色素c之间的序列差异如何完善该开关，当蛋白质不与心磷脂相互作用时关闭，当蛋白质与心磷脂相互作用时打开。该项目为两名研究生提供分子生物学方法、蛋白质表达、生物物理方法、结构生物学和单分子方法方面的培训，使他们能够发展成为有生产力的科学家，从而促进我们科学基础设施的发展。该项目还为MT . Missoula的一家科学博物馆开发了视觉上令人兴奋的教育材料，展示了蛋白质是如何发挥其功能的，目的是激励年轻人从事科学事业。本项目使用合理设计的细胞色素c变体来评估控制进入蛋白质过氧化物酶活性构象的序列决定因子。利用吸光度、荧光和圆二色光谱、单分子方法和x射线晶体学，采用平衡和停止流动动力学方法，探测心磷脂膜存在和不存在时细胞色素c变异体的结构动力学。在心磷脂膜存在和不存在的情况下评估过氧化物酶活性，以将变异依赖的构象和动态特性与过氧化物酶活性变化的光谱特征联系起来。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.