CYT STRUCTURE-FUNCTION VIA SITE-DIRECTED MUTAGENESIS

尽管自细胞色素c被重新发现以来，人们对其进行了广泛的研究 凯林在20世纪20年代，关于以何种方式 其功能特性由其结构决定。这些问题 仍然在很大程度上是由于化学修饰技术无法 从结构歧义看多种氨基酸残基 这在对来自不同物种的蛋白质的比较研究中总是发生的 物种。分子遗传学的最新进展现在提供了一种新的方法 通过应用 寡聚脱氧核糖核苷酸定点突变。这种方法， 原则上，允许替换 蛋白质序列与任何其他自然存在的氨基酸残基，如果 对编码该蛋白质的基因进行功能克隆是可行的。 我们实验室以前的工作已经或事实上发展了所有的 与这项提议相关的遗传和物理技术。这 经验现在将被引导到准备和表征 五种突变酵母异L细胞色素c的功能特性 我们根据其功能特点对其进行分类 它们被设计用来探测的蛋白质。这些类别的突变体包括 轴向配体突变体，血红素硫醚键突变体，氢键 突变体、氧化还原伙伴识别突变体和高pH转变突变体。 我们实验室的初步研究表明，这些技术 可以很容易地从20种L酵母培养物中产生15-30毫克突变蛋白 并证明了通过这些技术产生的突变细胞色素 为研究细胞色素结构对细胞毒性的影响提供了有力的手段 蛋白质的功能。综合起来，报告的初步结果 毫不含糊地证明 建议的工作。

Although cytochrome c has been studied extensively since its rediscovery by Kelin in the 1920s, many uncertainties remain regarding the manner in which its functional properties are determined by its structure. These questions remain largely from the inability of chemical modification techniques to probe many types of amino acid residues and from the structural ambiguities that invariably occur in comparative studies of proteins from different species. Recent advances in molecular genetics now provide a new method of addressing these concerns through the application of oligodexyribonucleotide-directed site-specific mutagenesis. This approach, in principle, permits the replacement of any residue in any position of the protein sequence with any other naturally occurring amino acid residue if a functional clone of the gene coding for the protein is available. Previous work in our laboratories has employed or, in fact, developed all of the genetic and physical techniques relevant to this proposal. This experience will now be directed to preparing and characterizing the functional properties of five types of mutant yeast iso-l-cytochromes c which we have classified on the basis of the functional features of the protein that they are designed to probe. These classes of mutants include axial ligand mutants, heme thioether linkage mutans, hydrogen-bonding mutants, redox partner recognition mutants, and high-pH transition mutants. Initial studies in our laboratories have demonstrated that these techniques can readily generate 15-30 mg of mutant protein from 20 L cultures of yeast and have demonstrated that mutant cytochromes produced by these techniques provide a powerful means for studying the effect of cytochrome structure on the function of the protein. Combined, the initial results reported unambiguously demonstrate the feasibility and potential productivity of the work proposed.