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 相互作用对蛋白质结构和功能很重要。 蛋白质折叠，获得最终的三维三级结构 一级氨基酸序列的结构，是最后一步 将 DNA 中编码的遗传信息转化为功能性信息 产品。 这种三级结构非常具体：a 的突变 单个蛋白质中的单个氨基酸残基可以引起转化或 消除原本健康细胞的活力。 许多不同的技术被用来研究这个 问题。 定点诱变已被用来确定 特定残基对于特定的折叠或功能的重要性 蛋白质。 定向随机诱变，定点诱变的一种形式 诱变，是一种使一个或多个残基发生突变的技术 在一次实验中分解成所有可能的氨基酸，这使得 有关残留物“可替代性”的更多信息，请参见 一度。 在另一种形式的诱变中，寡核苷酸定向 诱变，整个蛋白质片段 - 二级结构， 超二级结构或域 - 用单个修饰 寡核苷酸。 层次结构或模块化程度 可以通过此类实验来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定向 诱变 - iso-l-细胞色素 c，一种重要且高度保守的 酿酒酵母中的电子传递蛋白，In 为了确定对结构的影响或突变 该蛋白质的功能。 寡核苷酸定向诱变是 已经在该系统中使用，但是随机诱变技术 必须先开发。 那么，这项工作有四个具体目标：1） 继续寡核苷酸定向的环和螺旋诱变 Iso-l-细胞色素 c 以确定哪些区域在结构上或 功能上重要； 2) 光谱和动力学研究 体内和体外的特性或突变蛋白，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 用于在 Iso-l-细胞色素 c 中进行定向、随机诱变的系统； 4) 使用随机诱变进一步探测结构和功能 该蛋白质的关系。 定向、随机诱变的结果 寡核苷酸定向诱变之前尚未得到一致的研究 在这个系统中应用，但会导致更好的理解 参与折叠的特定残基或残基组 细胞色素c的功能；此外，推导出任何一般折叠规则 对于细胞色素c可能适用于其他蛋白质的折叠。