Evolution of Cytochrome c for Optimal Apoptotic Peroxidase Activity

程序性细胞死亡（细胞凋亡）是控制生物体内不同组织生长和发育的关键生化途径。细胞凋亡的一个重要的早期步骤是线粒体膜脂质，心磷脂，由活性氧物种，包括过氧化物的氧化。这种氧化活性充当有效的细胞凋亡开/关开关，由蛋白质细胞色素c的过氧化物酶活性促进。细胞色素c的固有过氧化物酶活性必须最小化，以免意外触发开关。有了这个奖项，化学部的生命过程化学项目正在资助布鲁斯E。蒙大拿大学的Bowler研究细胞色素c的氨基酸序列如何优化其在细胞凋亡中的过氧化物酶功能。这项资助旨在剖析酵母（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这种开关，当蛋白质不与心磷脂相互作用时关闭，当它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的培训，使他们能够发展成为富有成效的科学家，使我们的科学基础设施得以发展。该项目还为蒙大拿州米苏拉的一个科学博物馆开发了视觉上令人兴奋的教育材料，展示了蛋白质如何发挥其功能，旨在激励年轻人追求科学事业。该项目使用合理设计的细胞色素c变体来评估控制蛋白质过氧化物酶构象的序列决定因素。平衡和停流动力学的方法来探测在存在和不存在的心磷脂膜的细胞色素c变体的结构动力学，使用吸光度，荧光和圆二色谱，单分子方法和X-射线晶体学。在存在和不存在心磷脂膜的情况下评估过氧化物酶活性，以将过氧化物酶活性变化与光谱表征的变体依赖性构象和动力学性质相关联。该奖项反映了NSF的法定使命，并被认为值得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来支持。

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.