CHARACTERIZATION OF PROTEIN FOLDING INTERMEDIATES

根据这项提案开展的工作将集中在 非自然状态的表征和展开的路径 具有不同拓扑结构和二级结构的一系列蛋白质。 分子动力学方法和新发展的研究方法 将使用回转伞半径抽样进行调查 葡萄球菌无肌红蛋白片段B的非天然状态 蛋白A、溶菌酶和载脂蛋白α-乳清蛋白。的型号 可滴定侧链的质子化状态 代表无肌红蛋白Will蛋白的关键pH状态 有待开发和研究--体系在中性pH、pH 4和较低的pH值将被考虑。然后，这些系统将 使用平衡和非平衡技术进行研究 显式水溶液中的(T-JUMP)分子动力学。类似 将进行溶菌酶、载脂蛋白-α-乳清蛋白和 蛋白质A使用最近发展起来的半径技术 旋转伞采样，展开路径依次为 这些蛋白质系统(在显性溶剂中)在增量时 旋转半径的增加是由旋转半径的变化引起的 将检查回转半径偏置电势。这 途径，以及以中间值发展的中间体 回转半径，将与实验进行比较 确定的途径和中间体以及我们从 均衡和T跳跃模拟。这些活动最初的重点是 研究将描述小型企业的展开途径 三螺旋束蛋白，蛋白A，因为它的简化 拓扑学。这个系统的折叠路径目前是 用快速动力学方法和核磁共振氢气进行了实验研究 交换。

Work carried out under this proposal will focus on the characterization of non-native states and unfolding pathways for a series of proteins with varying topology and secondary structure. The methods of molecular dynamics and newly developed approaches of radius of gyration umbrella sampling will be used to investigate non-native states of apomyoglobin, fragment B of staphylococcal protein A, lysozyme and apo-alpha-lactalbumin. Models for the protonation states of titratable side chains which are representative of key pH states for the protein apomyoglobin will be developed and studied - models for the system at neutral pH, pH 4 and lower pH values will be considered. These systems will then be investigated using techniques of equilibrium and non-equilibrium (T-jump) molecular dynamics in explicit aqueous solvent. Similar studies will be carried out for lysozyme, apo-alpha-lactalbumin and protein A. Using recently developed techniques of radius of gyration umbrella sampling, the unfolding pathway followed by each of these protein systems (in explicit solvent) when incremental increases in the radius of gyration are induced by changes in the radius of gyration biasing potential will be examined. This pathway, and the intermediates which develop at intermediate values of radius of gyration, will be compared to experimentally determined pathways and intermediates as well as those we find from equilibrium and T-jump simulations. The initial focus of these studies will be to characterize the unfolding pathway for the small three helical bundle protein, protein A, because of its simplified topology. The folding pathway of this system is currently being studied experimentally using fast kinetic methods and NMR hydrogen exchange.