Kinetics of Early Events in Protein Folding

描述(由申请人提供)：许多小的蛋白质在形成的限速步骤之前显示出构象变化的证据 密集的原生结构。尽管进行了密集的研究，但我们对 这些早期折叠的物理性质和机械作用 中间体仍然不完整。特别是，人们对此知之甚少。 早期中间体的链拓扑结构和三级结构偏好， 它们对于理解折叠是如何启动和引导至关重要的 沿着富有成效的渠道。这个项目的一个主要目标是阐明 瞬变状态的结构特性和遇到的动能垒 在两个有代表性的模型蛋白折叠的早期阶段，蛋白G 和葡萄球菌核酸酶。折叠的初始阶段一直延伸到 将通过耦合先进的快速混合来探索微秒时间尺度 具有结构信息的构象探针的方法，如内在 和外在荧光探针，以及对单个酰胺氢的保护 来自核磁共振监测的溶剂交换。特定残留物的参与 以及稳定暂态的相互作用和折叠中的势垒 蛋白质G将通过结合这些动力学方法和 定点突变。结果将确定关键的结构特征 参与这个原型单域蛋白折叠的每个阶段。 对疏水星团形成的详细洞察，具体 荧光猝灭相互作用与长程距离分布 在折叠过程中，通过动力学分析得到葡萄球菌核酸酶 具有工程色氨酸残基和荧光能量的变体 转移方式。关于氢键形成的补充信息 折叠早期阶段的结构将通过组合 核磁共振检测氢交换的超快淬火流动法。这个 洞察这些蛋白质和其他不同蛋白质的快速折叠事件 结构性能将为测试提供坚实的实验基础 蛋白质折叠、结构预测的理论和计算模型 和从头开始的蛋白质设计。

DESCRIPTION (provided by applicant): Many small proteins show evidence for conformational changes prior to the rate-limiting step in the formation of the densely packed native structure. Despite intense study, our understanding of the physical properties and mechanistic role of these early folding intermediates remains incomplete. In particular, little is known about the chain topology and tertiary structural preferences in early intermediates, which are critical for understanding how folding is initiated and directed along productive channels. A major goal of this project is to elucidate the structural properties of the transient states and kinetic barriers encountered during early stages of folding of two representative model proteins, protein G and staphylococcal nuclease. Initial stages of folding extending well into the microsecond time scale will be explored by coupling advanced rapid mixing methods with structurally informative conformational probes, such as intrinsic and extrinsic fluorescence probes, and protection of individual amide hydrogens from solvent exchange monitored by NMR. The involvement of specific residues and interactions in stabilizing transient states and barriers in folding of protein G will be explored by combining these kinetic methods with site-directed mutagenesis. The results will identify key structural features involved in each stage of folding of this prototypic single-domain protein. Detailed insight into the formation of hydrophobic clusters, specific fluorescence quenching interactions and long-range distance distributions during folding of staphylococcal nuclease will be obtained by kinetic analysis of variants with engineered tryptophan residues and fluorescence energy transfer methods. Complementary information on the formation of hydrogen bonded structure during early stages of folding will be obtained by combining ultrarapid quenched-flow methods with NMR-detected hydrogen exchange. The insight into fast folding events for these and other proteins with diverse structural properties will provide a firm experimental basis for testing theoretical and computational models of protein folding, structure prediction and de novo protein design.