FOLDING AND DYNAMICS OF A MOLTEN GLOBULE

描述：本提案的长期目标是了解 蛋白质折叠的机制，一个新合成的 多肽采用其生物构象。 一般蛋白质折叠 中间体是熔融球，一种以致密为特征的物质， 二级结构的接近天然水平，以及缺乏刚性， 特殊的侧链包装。 研究得最好的熔融球之一是 α-乳白蛋白（α-LA）是一种小的双结构域蛋白。 的 丰富的现有知识，探测主干拓扑的能力 通过二硫键形成，以及重组变体的可用性 在非变性条件下是熔融球， 理想的模型系统，以了解这种中间体的功能， 蛋白质折叠 α-LA熔融球的螺旋结构域具有 天然的三级折叠，作为组织的支架， 多肽链的其余部分和搜索的起点 用于正确的侧链包装。 这项研究的具体目标是： (1)为了理解分子间的相互作用和信息， 一级序列，决定天然样三级折叠， α-LA熔融球。 (2)为了表征侧链动力学， 熔融球，并了解如何动态变化后，形成的 天然蛋白质 实现这些目标：（1）丙氨酸扫描诱变 和成对的丙氨酸取代将用于确定 每个侧链及其相互作用对特异性的贡献 形成类似天然的第三褶皱。 (2)NMR弛豫测量 选择性同位素标记的蛋白质将用于研究动力学 熔融球中的单个残留物， 从熔融小球到天然状态的α-LA的转变。 这些 这些研究将为理解这些疾病的分子基础提供基础。 由蛋白质错误折叠或聚集引起的人类疾病，以及 合理设计具有特定医学应用的蛋白质。

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.