CHARACTERIZATION OF PROTEIN FOLDING INTERMEDIATES

根据本建议开展的工作将侧重于 非天然状态和展开途径的表征， 具有不同拓扑结构和二级结构的一系列蛋白质。 分子动力学方法和近年来发展起来的分子动力学方法， 将使用回转半径伞抽样进行调查 葡萄球菌脱辅基肌红蛋白片段B的非天然状态 蛋白A、溶菌酶和脱辅基-α-乳白蛋白。模型的 可滴定侧链的质子化状态， 蛋白质脱辅基肌红蛋白的关键pH状态的代表将 开发和研究-模型的系统在中性pH值，pH值 4和更低的pH值将被考虑。这些系统将 使用平衡和非平衡技术进行研究 （T-跳跃）明确的水溶剂中的分子动力学。类似 将对溶菌酶、apo-α-乳白蛋白和 蛋白a使用最近开发的半径技术， 旋转伞采样，展开途径，其次是每个 这些蛋白质系统（在明确的溶剂）时，增量 回转半径的增加是由 将检查回转半径偏置电位。这 途径，以及在中间值处发展的中间体 的回转半径，将比较实验 确定的途径和中间体以及我们从中发现的那些 平衡和T跳跃模拟。这些最初的焦点 研究将是表征小分子的展开途径， 三螺旋束蛋白，蛋白A，因为它的简化， topology.该系统的折叠途径目前正在研究中。 实验研究使用快速动力学方法和核磁共振氢 交易所

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.