STRUCTURAL MODULARITY & PROTEIN FUNCTION IN CYTOCHROME C

结构模块化

• 批准号: 3468221
• 负责人: JACQUELYN Su FETROW
• 金额: $ 10.36万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468221
• 关键词: Saccharomyces; X ray crystallography; circular dichroism; conformation; cytochrome c; electron transport; gene deletion mutation; genetic manipulation; nuclear magnetic resonance spectroscopy; polarography; potentiometry; protein engineering; protein folding; protein sequence; protein structure function; site directed mutagenesis; ultraviolet spectrometry; western blottings

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.

这项工作的长期目标是了解 对蛋白质结构和功能很重要的相互作用。 蛋白质折叠，最终三维三级的获取 来自原代氨基酸序列的结构是最后一步 将DNA中编码的遗传信息转换为功能 产品。 该三级结构非常具体： 单蛋白中的单个氨基酸残基会导致转化或 消除原本健康的细胞的活力。 许多不同的技术正在用于研究此事 问题。 定点诱变已用于确定 特定残基在特定的折叠或功能上的重要性 蛋白质。 定向的，随机的诱变，一种形式的位置定向 诱变，是一种突变残基或残基突变的技术 在一个实验中进入所有可能的氨基酸，这允许很多 有关残留物的“替代性”的更多信息 一度。 在另一种形式的诱变形式中，寡核苷酸导向 诱变，全蛋白段 - 二级结构， 超少结构或域 - 通过一个单一修改 寡核苷酸。 分层结构或模块化程度 可以从这种实验中确定蛋白质中的存在。 本提案中描述的研究将应用这些 技术 - 定向，随机诱变和寡核苷酸导向 诱变 - 对ISO-L-CytoChrome C，一个重要且高度保守的 酿酒酵母中的电子传输蛋白在酿酒酵母中 为了确定结构的影响或突变， 该蛋白质的功能。 寡核苷酸指导的诱变为 已经在该系统中使用了，但是随机诱变的技术 必须首先开发。 因此，这项工作有四个具体目标：1） 继续在寡核苷酸指导的环路和螺旋中的诱变 ISO-L-CytoChrome C确定哪些区域在结构上或 在功能上很重要； 2）研究光谱和动力学 特性或体内和体外的突变蛋白，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 用于执行ISO-L-环形C中随机诱变的系统； 4）使用随机诱变来进一步探测结构和功能 该蛋白质的关系。 定向，随机诱变和 寡核苷酸指导的诱变之前尚未始终如一 应用于该系统，但会更好地理解 折叠中涉及的特定残基或一组残留物， 细胞色素c的功能；此外，推导的任何一般折叠规则 对于细胞色素C，可能适用于其他蛋白质的折叠。