MUTATIONAL STUDY OF THE MECHANISM OF PROTEIN FOLDING

蛋白质折叠机制的突变研究

• 批准号: 3301088
• 负责人: DAVID P GOLDENBERG
• 金额: $ 12.99万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301088
• 关键词: Escherichia coli; X ray crystallography; chemical stability; chemical substitution; conformation; disulfide bond; genetic manipulation; high performance liquid chromatography; hydrogen bond; protein engineering; protein folding; protein sequence; protein structure; site directed mutagenesis; synthetic nucleotide; thermodynamics; trypsin inhibitors

The mechanism of folding of a small, well-characterized protein, bovine pancreatic trypsin inhibitor (BPTI), will be studied by testing the effects of amino acid substitutions on the stabilities of folding intermediates and oh the stability and conformation of the native protein. this project is directed towards the long-term goal of understanding how the three-dimensional structures of globular proteins are specified by their amino acid sequences and how changes in the amino acid sequence may alter the properties of the native protein or the process of forming it. Many diseases involving defects in protein function may arise because of mutations that prevent proteins from acquiring the correct three-dimensional structures required to interact specifically with other molecules. Modified forms of BPTI will be produced using in vitro mutagenesis and recombinant DNA methods. A cloned gene coding for BPTI will be modified using synthetic oligonucleotides and used to direct the synthesis of mutant proteins in Escherichia coli. The disulfide-coupled folding reactions of the mutant proteins will be studied by chemically trapping and characterizing disulfide-bonded intermediates in folding and unfolding. To allow quantitative comparisons of he mutant and wild-type proteins, the relative stabilities of the native , unfolded and intermediate state will be measured, as well as their rates of interconversion. Some of the mutations to be studied ar site-directed changes designed to test the roles of specific residues and interactions in stabilizing the native protein and determining the pathway of folding. These site- directed changes are intended to disrupt interactions present in the native protein, including hydrogen bonds, salt bridges and packing interactions. Other mutants to be studied were identified amount randomly mutagenized clones using a genetic screen to isolate mutants with altered folding intermediate as well as in the native protein is expected to alter the stability of both species similarly. From the observed effects of the mutations on the relative stabilities of the different species and their kinetics of interconversion, the roles of he altered residues at the various stages of folding will be inferred.

折叠小蛋白质的折叠机制 胰腺胰蛋白酶抑制剂（BPTI）将通过测试研究 氨基酸取代对折叠稳定性的影响 中间人和哦，本地的稳定性和构象 蛋白质。 该项目针对的是 了解球状蛋白的三维结构 由它们的氨基酸序列以及氨基的变化方式指定 酸序列可能会改变天然蛋白或 形成它的过程。 许多涉及蛋白质缺陷的疾病 由于突变可防止蛋白质的突变，可能会产生功能 获得相互作用所需的正确的三维结构 特别是其他分子。 使用体外诱变和 重组DNA方法。 将修改编码BPTI的克隆基因 使用合成寡核苷酸，用于指导合成 大肠杆菌中的突变蛋白。 二硫键耦合折叠 突变蛋白的反应将通过化学诱捕研究 并表征二硫键键入的中间体在折叠和 展开。 允许对He突变体和野生型的定量比较 蛋白质，当地人的相对稳定性，展开和 将测量中间状态，及其率 互换。 某些要研究的突变旨在 测试特定残基和相互作用在稳定中的作用 天然蛋白质并确定折叠途径。 这些站点 - 定向更改旨在破坏存在 天然蛋白质，包括氢键，盐桥和包装 互动。 确定要研究的其他突变体数量 使用遗传筛选分离突变体的随机诱变克隆 随着折叠中间体以及本地蛋白质的变化 预计将同样改变这两种物种的稳定性。 来自 观察到突变对相对稳定性的影响 不同的物种及其相互转换的动力学，他的角色 将推断在折叠各个阶段的残留物改变。