Monomers and Dimers of Cytochrome c in Apoptotic Peroxidase Activity

化学部生命过程化学项目的这一奖项将资助蒙大拿大学的Bruce E.Bowler博士研究一种名为细胞色素c的蛋白质三维结构的变化如何改变其功能，从一种移动电子的蛋白质转变为一种加速膜脂(脂肪)与过氧化氢反应的蛋白质。后者，即所谓的过氧化物酶活性，在重要的生化途径中提供了早期信号。该项目还将为本科生和研究生提供广泛的结构、生化和生物物理方法方面的培训。作为该项目的一部分，鲍勒博士和他的学生正在开发实践课程材料，在资源匮乏的环境中有效，以加强部落学校的STEM教育，并与米苏拉的光谱科学中心合作。开展这项研究的假设源于与洗涤剂Cymal-6结合的酵母iso-1-细胞色素c的一种新的X射线衍射结构。在Cymal-6存在下，iso-1-细胞色素c结晶为结构域互换的二聚体，与cymal-6结合在口袋中，将其碳氢链放置在血红素铁旁边。鲍勒博士的实验室正在通过测量单体和二聚体细胞色素c的过氧化物酶活性，确定心磷脂纳米盘催化形成二聚体的能力，以及测试脂质类似物稳定二聚体的能力，来验证这种二聚体结构对过氧化物酶活性重要的假设。对不同脂类类似物的结晶和结构研究也在尝试，以更全面地确定二聚体的脂结合口袋。这项工作是用三种进化上不同的细胞色素c进行的，它们是酵母、蜘蛛猴和人类。还在研究脂结合口袋附近两个表面环的突变对结合口袋的可及性和细胞色素c的过氧化物酶活性的影响，以确定从酵母到人类的进化如何优化细胞色素c的过氧化物酶活性。最后，细胞色素c的二聚体结构表明，广泛的赖氨酸残基控制着细胞色素c与心磷脂的静电结合。细胞色素c变异体与这些赖氨酸的结合研究正在进行中，以确定细胞色素c表面的静电对接位置的程度。

This award from the Chemistry of Life Processes Program within the Chemistry Division is funding Dr. Bruce E. Bowler from the University of Montana to investigate how changes in the three-dimensional structure of a protein known as cytochrome c changes its function from a protein that moves electron around to one that speeds up the reaction of membrane lipids (fats) with hydrogen peroxide. This latter, so-called, peroxidase activity provides an early signal in important biochemical pathways. This project will also provide training for undergraduate student and graduate students in a broad array of structural, biochemical and biophysical methods. As part of this project, Dr. Bowler and his students are developing hands-on curricular materials, effective in low resource environments, to enhance STEM education at tribal schools in collaboration with the spectrUM science center in Missoula. The hypotheses for the research to be carried out derive from a novel X-ray diffraction structure of yeast iso-1-cytochrome c bound to the detergent CYMAL-6. In the presence of CYMAL-6, iso-1-cytochrome c crystallizes as a domain-swapped dimer with CYMAL-6 bound in a pocket placing its hydrocarbon chain next to the heme iron. Dr. Bowler's lab is testing the hypothesis that this dimeric structure is important for peroxidase activity by measuring the peroxidase activity of monomeric versus dimeric cytochrome c, determining the ability of cardiolipin nanodiscs to catalyze the formation of dimer, and also testing the ability of lipid analogs to stabilize the dimer. Crystallization and structural studies with different lipid analogs also is being attempted to more fully define the lipid binding pocket of the dimer. The work is being carried out with three evolutionally diverse forms of cytochrome c, those of yeast, spider monkey and human. The effects of mutations to two surface loops near the lipid binding pocket, on the accessibility of the binding pocket and the peroxidase activity of cytochrome c also is being investigated to determine how evolution from yeast to humans has optimized cytochrome c for peroxidase activity. Finally the dimeric structure of cytochrome c indicates that a broad group of lysine residues control the electrostatic binding of cytochrome c to cardiolipin. Binding studies on cytochrome c variants with these lysines eliminated is being conducted to determine the extent of the electrostatic docking site on the surface of cytochrome c.