STRUCTURAL MODULARITY & PROTEIN FUNCTION IN CYTOCHROME C

这项工作的长期目标是了解 这些相互作用对蛋白质的结构和功能非常重要。 蛋白质折叠，获得最终的三维三级结构 从一级氨基酸序列中合成结构，是 将编码在DNA中的遗传信息翻译成功能性的 产品 这种三级结构是非常特殊的： 单个蛋白质中单个氨基酸残基可引起转化或 消除健康细胞的活力。 许多不同的技术正在被用来研究这个 问题. 定点诱变已被用于确定 特定残基对于特定蛋白质的折叠或功能的重要性 蛋白 定向突变，随机突变，定点突变的一种 诱变是一种技术，其中一个或多个残基突变 在一个实验中转化为所有可能的氨基酸， 关于残留物的“可替换性”的更多信息 一次性 在另一种形式的诱变中， 诱变，整个蛋白质片段-二级结构， 超二级结构，或结构域-用单个 寡核苷酸。 等级结构或模块化程度 蛋白质中存在的蛋白质可以从这种类型的实验中确定。 本提案中描述的研究将应用这些 技术-定向、随机诱变和阿托伐他汀定向 诱变-异-L-细胞色素C，一个重要的和高度保守的 电子传递蛋白在酿酒酵母中， 为了确定对结构的影响或突变， 这种蛋白质的功能。 寡核苷酸定向诱变是 已经在这个系统中使用，但是随机诱变技术 必须首先开发。 因此，这项工作有四个具体目标：1） 环和螺旋的连续寡核苷酸定向诱变 异-l-细胞色素c，以确定哪些区域在结构上或 2）研究了光谱和动力学 性质或突变蛋白在体内和体外，以确定如何 每个突变都会影响蛋白质的结构和功能; 3）开发一个 用于在异-l-细胞色素c中进行定向随机诱变的系统; 4）利用随机突变进一步探索结构和功能 这种蛋白质的关系。 定向、随机诱变和 阿替西肽定向诱变以前并不一致， 在这个系统中应用，但将导致更好地了解 参与折叠的特定残基或残基组， 细胞色素c的功能;此外，任何一般的折叠规则推导 细胞色素c的折叠可能适用于其他蛋白质的折叠。

The long-term objective of this work is to understand the interactions which are important for protein structure and function. Protein folding, the acquisition of a final, three-dimensional tertiary structure from a primary amino acid sequence, is the final step in translating the genetic message encoded in the DNA into a functional product. This tertiary structure is very specific: the mutation of a single amino acid residue in a single protein can cause transformation or eliminate viability of an otherwise healthy cell. Many different technologies are being used to study this problem. Site-directed mutagenesis has been used to determine the importance of specific residues for the folding or function of a specific protein. Directed, random mutagenesis, one form of site-directed mutagenesis, is a technique in which a residue or residues are mutated into all possible amino acids In a single experiment, which allows much more Information about a residue's "replaceability" to be determined at one time. In another form of mutagenesis, oligonucleotide-directed mutagenesis, whole protein segments - secondary structures, supersecondary structures, or domains - are modified with a single oligonucleotide. The degree of hierarchical structure or modularity present In a protein can be determined from this type of experiment. The research described in this proposal will apply these techniques - directed, random mutagenesis and oligonucleotide-directed mutagenesis - to iso-l-cytochrome c, an important and highly conserved electron transport protein in the yeast Saccharomyces cerevisiae, In order to determine the affect or the mutations on the structure and function of this protein. Oligonucleotide-directed mutagenesis is already in use in this system, but the techniques of random mutagenesis must be first developed. This work, then, has four specific goals: 1) To continue oligonucleotide-directed mutagenesis of loops and helices In Iso-l-cytochrome c to determine which regions are structurally or functionally important; 2) To study the spectroscopic and kinetic properties or the mutant proteins in vivo and in vitro to determine how each mutation affects the protein structure and function; 3) To develop a system for performing directed, random mutagenesis in Iso-l-cytochrome c; and 4) To use random mutagenesis to further probe structure and function relationships in this protein. The results of directed, random mutagenesis and oligonucleotide-directed mutagenesis have not before been consistently applied in this system, but will lead to a better understanding of the specific residues or groups of residues involved in the folding and function of cytochrome c; furthermore, any general folding rules deduced for cytochrome c may be applicable to the folding of other proteins.