Structure Function Studies of Cytochrome c

这个交叉学科项目由数学和物理科学局的无机、生物无机和有机金属化学计划和生物科学局的生物物理学计划支持，结合了化学家和分子生物学家在阐明细胞色素c结构功能关系方面的专业知识。研究的目的是：1)检测，在体外，血红素环境中的突变对细胞色素的氧化还原电位和电子转移活性的影响，并将体外观察到的影响与体内细胞色素的活性进行比较，以及2)使用动力学和热力学方法来确定突变细胞色素的稳定性，最终目的是确定蛋白质稳定性如何与细胞的蛋白质表达水平相关联。重组DNA程序将被用来从酿酒酵母中获得突变的细胞色素，这将用于这些研究。使用血红素发色团的光谱响应来估计细胞中存在的细胞色素的数量。这项研究的目的是加深我们对生物分子，特别是金属蛋白的结构变化影响其生物学功能的方式的理解。由于很难在体内直接观察生物分子，最初的研究将在体外进行，然后可以在体内研究的行为，如细胞产生蛋白质的速度，将与体外测量相关联。

This interdisciplinary project supported by the Inorganic, Bioinorganic and Organometallic Chemistry Program in the Mathematical and Physical Sciences Directorate and the Biophysics program in the Biological Sciences Directorate combines the expertise of chemists and molecular biologists in the elucidation of structure function relationships in cytochrome c. The aims of the research are: 1) to examine, in vitro, the effects of mutations within the heme environment on the redox potential and electron transfer reactivity of the cytochrome, and to compare the effects observed in vitro with the in vivo reactivity of the cytochrome, and 2) to determine the stabilities of the mutant cytochromes using both kinetic and thermodynamic methods with the ultimate objective of determining how protein stability can be linked to the level of protein expression of the cell. Recombinant DNA procedures will be used to obtain the mutant cytochromes from the yeast Saccharomyces cerevisiae that will be used in these studies. Estimation of the amount of cyctochrome present in cells will be done using the spectral response of the heme chromaphore. %%% The objective of this research is to further our understanding of the ways in which variations in the structure of biomolecules, and that of metalloproteins in particular, affect their biological function. Because of the difficulty of making direct observations on biological molecules in vivo, studies will initially be made in vitro and then behavior that can be studied in vivo, such as the rate of protein production by the cell, will be correlated with the in vitro measurements.