MUTATIONAL STUDY OF PROTEIN FOLDING AND DYNAMICS

蛋白质折叠和动力学的突变研究

• 批准号: 6385930
• 负责人: DAVID P GOLDENBERG
• 金额: $ 24.92万
• 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 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385930
• 关键词: chemical stability; conformation; disulfide bond; high performance liquid chromatography; intermolecular interaction; molecular dynamics; molecular rearrangement; mutant; neurotoxins; nuclear magnetic resonance spectroscopy; protein folding; protein sequence; structural biology; thermodynamics; trypsin inhibitors

The long term goal of this project is to develop a quantitative understanding of the roles of individual amino acid residues and interactions in directing the folding of globular proteins. Mutational analysis, in conjunction with biochemical experiments and high- resolution NMR spectroscopy, will be used to study intermediates in the disulfide-coupled refolding of two small proteins, bovine pancreatic trypsin inhibitor (BPTI) and an omega-conotoxin. The major disulfide-bonded intermediates in the refolding of BPTI are presently among the best characterized for any protein. In order to determine what interactions and structural features favor the formation of the different intermediates, and how these contributions change as folding proceeds, the effects of sequence changes on the stabilities and kinetic roles of the intermediates will be measured. The modifications to be studied are designed either to eliminate side-chain hydrogen bonds, force changes to the native backbone conformation, or alter the covalent connectivity of the backbone. To obtain a more detailed understanding of how individual residues influence polypeptide conformation and dynamics, NMR spectroscopy will be used to examine the effects of amino acid replacements on native BPTI and an intermediate that lacks one of the three disulfides. The omega-conotoxins are unusually small disulfide-bonded proteins that function as antagonists of voltage-gated Ca2+ channels. In spite of their small size, 25-30 residues, they are able to form their three disulfides and fold into well-defined three-dimensional structures. To test for the presence and roles of stabilizing interactions early in folding, the relative stabilities of all of the possible one-disulfide intermediates will be determined, and the influence of each of the native disulfides on forming a second native disulfide will be measured. NMR spectroscopy will be used to study the flexibility and conformations of the three species containing two of the native disulfides, and mutational analysis will be used to asses the contributions of selected non-Cys residues to the stability of the folded conformation. The results of this project will contribute to the fundamental knowledge necessary to realize the health care potentials offered by the revolutionary developments in genetic analysis and manipulation. Possible applications of these results include the design of proteins with enhanced stabilities or novel activities and improved understanding of how naturally occurring mutations lead to disease states by preventing protein folding or altering the structure or dynamics of folded proteins.

该项目的长期目标是开发定量 了解单个氨基酸残基和 指导球状蛋白质折叠时的相互作用。突变 分析，结合生化实验和高 分辨率NMR光谱法将用于研究中间体 两种小蛋白的二硫键偶联重折叠，牛胰腺 胰蛋白酶抑制剂（BPTI）和omega-conotoxin。 BPTI重新折叠的主要二硫键中间体是 目前是任何蛋白质的最佳特征。 为了 确定哪些相互作用和结构特征有利于形成 不同的中间体，以及这些贡献如何变化 折叠的过程，序列的影响对稳定性的变化和 将测量中间体的动力学作用。 修改 待研究的设计是为了消除侧链氢 键，力变为天然骨干构象或改变 主链的共价连通性。 获得更详细的 了解单个残留物如何影响多肽 构象和动力学，NMR光谱将用于检查 氨基酸替代对天然BPTI和中间体的影响 缺少三个二硫化物之一。 欧米茄 - 连毒素是异常小的二硫键蛋白， 充当电压门控Ca2+通道的拮抗剂。 尽管 它们的小尺寸，25-30个残留物，能够形成三个 二硫结构二维结构。 到 测试早期稳定相互作用的存在和作用 折叠，所有可能的一硫化物的相对稳定性 将确定中间体，每个中间体的影响 将测量形成第二个天然二硫化物的天然二硫化物。 NMR光谱法将用于研究灵活性和构象 在包含两个本地二硫化物的三个物种中， 突变分析将用于评估选定的贡献 折叠构象稳定性的非Cys残基。 该项目的结果将有助于基本知识 实现实现医疗潜力的必要条件 遗传分析和操纵中的革命发展。 这些结果的可能应用包括蛋白质的设计 具有增强的稳定性或新颖的活动，并提高了理解 关于自然发生的突变如何导致疾病状态 防止蛋白质折叠或改变的结构或动力学 折叠蛋白。