Evolution of Cytochrome c for Optimal Apoptotic Peroxidase Activity

细胞色素 c 的进化以获得最佳的凋亡过氧化物酶活性

• 批准号: 1904895
• 负责人: Bruce Bowler
• 金额: $ 39万
• 依托单位: University of Montana
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1904895&HistoricalAwards=false
• 关键词: Evolution; Cytochrome; Optimal; Apoptotic; Peroxidase

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.

细胞程序性死亡(细胞凋亡)是控制生物体内不同组织生长发育的关键生化途径。细胞凋亡的一个重要早期步骤是线粒体膜脂心磷脂被包括过氧化氢在内的活性氧物种氧化。蛋白质细胞色素c的过氧化物酶活性促进了这种氧化活性，其作用是有效的凋亡开/关开关。细胞色素c固有的过氧化物酶活性必须最小化，以免意外地触发开关。该奖项由化学部生命过程化学项目资助蒙大拿大学的Bruce E.Bowler博士研究细胞色素c的氨基酸序列如何针对其在细胞凋亡中的过氧化物酶功能进行优化。这项资助旨在剖析酵母(无凋亡途径)和人类(功能凋亡途径)细胞色素c之间的序列差异如何完善这一开关，使其在蛋白质与心磷脂不相互作用时关闭，当蛋白质与心磷脂相互作用时打开。这个项目为两名研究生提供了分子生物学方法、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多产的科学家，使我们的科学基础设施得以发展。该项目还为密苏拉的一家科学博物馆开发了视觉上令人兴奋的教育材料，展示了蛋白质如何执行其功能，目的是激励年轻人从事科学事业。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活性构象的序列决定因素。采用平衡动力学和停流动力学方法，利用吸光度、荧光光谱、圆二色谱、单分子方法和X射线结晶学研究了有无心磷脂膜时细胞色素c异构体的结构动力学。过氧化物酶活性是在存在和不存在心磷脂膜的情况下进行评估的，以将光谱特征为过氧化物酶活性变化的变异依赖构象和动态性质联系起来。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis

人细胞色素 c 结构域交换二聚体促进内在细胞凋亡的严格调节

• DOI: 10.1021/acs.biochem.0c00326
• 发表时间: 2020
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Steele, Harmen B.;Elmer-Dixon, Margaret M.;Rogan, James T.;Ross, J. B.;Bowler, Bruce E.
• 通讯作者: Bowler, Bruce E.

Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c

脯氨酸含量和组氨酸连接对 β-loop D 动力学和 iso-1-细胞色素 c 过氧化物酶活性的影响

• DOI: 10.1016/j.jinorgbio.2023.112474
• 发表时间: 2024
• 期刊: Journal of Inorganic Biochemistry
• 影响因子: 3.9
• 作者: Martin, William J.;McClelland, Levi J.;Nold, Shiloh M.;Boshae, Kassandra L.;Bowler, Bruce E.
• 通讯作者: Bowler, Bruce E.

Curvature-Dependent Binding of Cytochrome c to Cardiolipin

• DOI: 10.1021/jacs.0c07301
• 发表时间: 2020-11-18
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Elmer-Dixon, Margaret M.;Xie, Ziqing;Bowler, Bruce E.
• 通讯作者: Bowler, Bruce E.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST

• DOI: 10.1021/jacs.3c04966
• 发表时间: 2023-10-10
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Rothfuss，Michael T.;Becht，Dustin C.;Bowler，Bruce E.
• 通讯作者: Bowler，Bruce E.