PHYSICAL AND IMMUNOLOGICAL STUDIES OF PROTEIN FOLDING

该项目的总体目标是阐明在 多肽的初级氨基酸序列决定了次要和次要氨基酸序列 这些多肽的三级构象及其折叠机制 这一点已经实现了。这将增加我们对 蛋白质在体内发挥作用的机制及其如何失灵 在某些疾病状态下。它也是我们未来设计和开发能力的关键 对蛋白质进行基因工程以达到预期目的。最初的目标是 确定20个或更少残基的氨基酸序列 在水溶液中形成α-螺旋。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 位于不同位置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。优势所在 组氨酸侧链与螺旋偶极相互作用的研究 应该直接反映在组氨酸的PK位移上。第二阶段 将利用葡萄球菌核酸酶，这种酶对 由于三级构象缺少二硫键而进行的研究。 我们计划在C.B.Anfinsen和他的同事们的工作基础上再接再厉 用多克隆血清检测链球菌核酸酶的构象状态 碎片。然而，我们将使用单抗，从而消除 与多克隆血清相关的问题。我们还将提供一个 定点突变产生的链球菌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的特异性和 蛋白质折叠机制。

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.