MUTATIONAL STUDY OF PROTEIN FOLDING

蛋白质折叠的突变研究

• 批准号: 2181419
• 负责人: DAVID P GOLDENBERG
• 金额: $ 16.91万
• 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 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181419
• 关键词: chemical stability; conformation; disulfide bond; gel electrophoresis; high performance liquid chromatography; hydrogen bond; hydropathy; intermolecular interaction; molecular dynamics; molecular rearrangement; mutant; nuclear magnetic resonance spectroscopy; protein engineering; protein folding; protein sequence; protein structure; site directed mutagenesis; structural biology; temperature; thermodynamics; trypsin inhibitors

The long term goal of this project is to develop a quantitative understanding of how individual interactions contribute cooperatively to conformational stability in both fully folded proteins and in partially folded intermediates that determine the mechanism of folding. Amino acid replacements will be used to probe the roles of individual residues in the refolding of bovine pancreatic trypsin inhibitor (BPTI), a small protein for which the folding pathway has been studied in great detail. Disulfide- bonded intermediates in the refolding of this protein contain much of the structure found in the native protein, but the extent and stability of this structure varies among the intermediates. Measuring the effects of amino acid replacements on the stabilities of the different intermediates will provide a uniquely detailed picture of how the contributions of individual residues are influenced by their environment within the protein at different stages of folding. Genetically modified BPTI variants will be produced in Escherichia coli and will be studied by chemically trapping, isolating and characterizing the disulfide-bonded intermediates, using techniques previously developed for the study of the wild-type protein. One of the major goals of this project is to measure the effects of substitutions on the kinetics and equilibria for forming the intermediates that contain only a single disulfide and represent an early stage of folding. Comparing the effects of substitutions on these intermediates with the effects of the same substitutions on the stability of the native protein will identify the major interactions that stabilize the intermediates and will indicate whether these interactions are as stable in the intermediates as they are in the fully folded protein. The second major goal is to learn how the stability of a single interaction in the native protein, a disulfide bond, is influenced by its environment For this purpose, the effects of substitutions on the last step of the pathway, the formation of a disulfide in the otherwise native protein, will be studied. Single amino acid replacements can alter the free energy change for this reaction by as much as 5 kcal/mol. To determine the structural basis of these effects, high resolution NMR will be used to study the structures and dynamics of mutant proteins with and without the disulfide. In addition, the roles of steric and entropic factors in determining disulfide stability will be probed by measuring the effects of multiple amino acid replacements and temperature on the equilibria and kinetics of the reaction. The results of this project will help provide the fundamental knowledge necessary to fully realize the health care potentials offered by the revolutionary developments in genetic analysis and manipulation. Possible applications of this knowledge include the design of proteins with enhanced stabilities or novel activities and improved understanding of how naturally occurring mutations lead to disease states by altering protein structure and dynamics.

该项目的长期目标是开发定量 了解个人互动如何合作 完全折叠蛋白和部分折叠蛋白的构象稳定性 折叠中间体，确定折叠机理。氨基酸 替换将用于探测单个残基在 牛胰腺胰蛋白酶抑制剂（BPTI）的再折叠，一种小蛋白 为此，对折叠路径进行了详细的研究。二硫键 粘合的中间体在该蛋白质的重新折叠中包含大部分 在天然蛋白质中发现的结构，但 这种结构在中间体之间有所不同。测量 氨基酸替代不同中间体的稳定性 将提供一个独特的详细图片，说明如何贡献 单个残留物受蛋白质内环境的影响 在折叠的不同阶段。转基因的BPTI变体将是 在大肠杆菌中产生，将通过化学诱捕研究， 使用二硫键键合中间体隔离和表征 以前用于研究野生型蛋白的技术。 该项目的主要目标之一是衡量 动力学和平衡的替换以形成中间体 仅包含一个二硫化物，代表了 折叠式的。比较取代对这些中间体的影响 具有相同替代对天然稳定性的影响 蛋白质将确定稳定的主要相互作用 中间体并将指示这些相互作用是否稳定 在中间体中，它们在完全折叠的蛋白质中。第二个 主要目标是了解单个交互的稳定性如何 天然蛋白是一种二硫键，受其环境的影响 此目的，替换对 途径，在原本天然蛋白质中形成二硫化物， 将被研究。单个氨基酸替代可以改变自由能 对该反应的变化多达5 kcal/mol。确定 这些影响的结构基础，高分辨率NMR将用于 研究有或没有的突变蛋白的结构和动力学 二硫化物。另外，空间和熵因子在 确定二硫化物稳定性将通过测量 在平衡和 反应动力学。 该项目的结果将有助于提供基本知识 充分意识到充分实现的医疗保健潜力所必需的 遗传分析和操纵中的革命发展。可能的 此知识的应用包括与 增强的稳定性或新颖的活动，并提高了对如何的理解 自然发生的突变通过改变蛋白质导致疾病状态 结构和动态。