FOLDING AND DYNAMICS OF A MOLTEN GLOBULE

描述：这项建议的长期目标是了解 蛋白质折叠的机制，一种新合成的蛋白质的过程 多肽采用其生物构象。一种通用的蛋白质折叠 中间是熔融球体，这是一种以致密为特征的物种， 接近原生水平的二级结构，没有僵硬的， 特殊的侧链包装。研究得最好的熔球之一是 α-乳清蛋白(α-LA)，一种小的双域蛋白。这个 丰富的现有知识，能够探测主干拓扑 通过二硫键的形成和重组变异体的可用性 在非变性条件下是熔融的球状物质使α-LA成为 理解这一中间体功能的理想模型系统 蛋白质折叠。α-LA熔融球的螺旋结构域具有 原住民式的第三折叠，作为组织 多肽链的其余部分和搜索的起点 正确的侧链包装。这项研究的具体目的是： (1)了解分子间的相互作用和信息 决定类天然第三折叠的初级序列 阿尔法-LA熔融球体。(2)表征侧链动力学。 熔化球体，并了解在形成球体时动力学如何变化 天然蛋白质。为了实现这些目标：(1)丙氨酸扫描诱变 并将使用成对的丙氨酸替代来确定 各侧链的贡献及其相互作用对特异性的影响 类原生第三系褶皱的形成。(2)核磁共振弛豫测量 关于选择性同位素标记的蛋白质将用于研究动力学 熔化的球体中的单个残留物，并理解 从熔融的球体转变为α-LA的天然状态。这些 研究将为理解分子基础提供基础。 由蛋白质错误折叠或聚集引起的人类疾病，以及 具有特定医疗应用的蛋白质的合理设计。

DESCRIPTION: The long term objective of this proposal is to understand the mechanism of protein folding, the process by which a newly synthesized polypeptide adopts its biological conformation. A general protein folding intermediate is the molten globule, a species characterized by compactness, near-native levels of secondary structure, and the absence of rigid, specific side chain packing. One of the best studied molten globules is that of alpha-lactalbumin (alpha-LA), a small two-domain protein. The richness of existing knowledge, the ability to probe the backbone topology by disulfide bond formation, and the availability of recombinant variants that are molten globules under non-denaturing conditions make alpha-LA an ideal model system for understanding the function of this intermediate in protein folding. The helical domain of alpha-LA molten globule has a native-like tertiary fold, which serves as a scaffold for organization of the rest of the polypeptide chain and a starting point from which to search for the correct side chain packing. The specific aims of this study are: (1) To understand the molecular interactions and the information in the primary sequence that determine the native-like tertiary fold in the alpha-LA molten globule. (2) To characterize the side chain dynamics in the molten globule and understand how the dynamics change upon formation of the native protein. To achieve these goals: (1) Alanine scanning mutagenesis and pairwise alanine substitutions will be used to determine the contribution of each side chain and its interaction to the specificity for formation of the native-like tertiary fold. (2) NMR relaxation measurements on selectively isotope labeled proteins will be used to study the dynamics of individual residues in the molten globule and to understand the transition from the molten globule to the native state of alpha-LA. These studies will provide foundations for understanding the molecular basis of human diseases caused by protein misfolding or aggregation, as well as for rational design of proteins with specific medical applications.