PHYSICAL AND IMMUNOLOGICAL STUDIES OF PROTEIN FOLDING

蛋白质折叠的物理和免疫学研究

• 批准号: 3085059
• 负责人: KAREN E STREHLOW
• 金额: $ 6.72万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3085059
• 关键词: antibody specificity; conformation; histidine; immunochemistry; monoclonal antibody; mutant; nuclear magnetic resonance spectroscopy

The overall aim of the project is to elucidate which elements in the primary amino acid sequence of polypeptides determine the secondary and tertiary conformation of these polypeptides and the folding mechanism by which this is accomplished. This will add to our understanding of the mechanisms by which proteins function in the body and how they malfunction in some disease states. It is also key to our future ability to design and genetically engineer proteins for desired purposes. An initial goal is to identify amino acid sequences of 20 or fewer residues that show substantial Alpha-helix formation in aqueous solution. This will enable us to make quantitative measurements of the helix dipole effect. We propose to use nuclear magnetic resonance to measure the effect of the helix dipole on the C2H resonance of a single histidine residue placed at various points along an (Ala-HO-propyl-L-Gln)N sequence with blocking end groups. The strength of the interaction between the histidine side chain and the helix dipole should be directly reflected in the pK shift of the histidine. Phase 2 will utilize staphylococcal nuclease which is particularly attractive for the study of tertiary conformation because of its lack of disulfide bonds. We plan to build upon work of C. B. Anfinsen and associates who used polyclonal sera to probe the conformational states of S. nuclease fragments. We will, however, use monoclonal antibodies, thus eliminating problems associated with polyclonal sera. We will also have available a library of S. nuclease mutants produced by site-directed mutagenesis and will be able to generate mutants to further probe antibody specificity and the protein folding mechanism.

该项目的总体目标是阐明 多肽的一级氨基酸序列决定了二级和 这些多肽的三级构象和折叠机制 这是完成的。 这将加深我们对 蛋白质在体内发挥作用的机制以及它们如何发生故障 在某些疾病状态下。 这也是我们未来设计和设计能力的关键 对蛋白质进行基因改造以达到所需目的。 最初的目标是 鉴定 20 个或更少残基的氨基酸序列，这些残基显示出显着的 水溶液中α-螺旋的形成。 这将使我们能够 螺旋偶极子效应的定量测量。 我们建议使用 核磁共振测量螺旋偶极子对 位于沿不同点的单个组氨酸残基的 C2H 共振 具有封闭端基的 (Ala-HO-丙基-L-Gln)N 序列。 实力 组氨酸侧链和螺旋偶极子之间的相互作用 应直接反映在组氨酸的 pK 变化上。 第二阶段 将利用葡萄球菌核酸酶，这对于 由于缺乏二硫键而对三级构象进行研究。 我们计划以 C. B. Anfinsen 及其同事的工作为基础，他们使用 多克隆血清探测链球菌核酸酶的构象状态 碎片。 然而，我们将使用单克隆抗体，从而消除 与多克隆血清相关的问题。 我们还将提供一个 通过定点诱变产生的 S. 核酸酶突变体文库和 将能够产生突变体以进一步探测抗体特异性 蛋白质折叠机制。