EARLY EVENTS IN PROTEIN FOLDING

蛋白质折叠成其天然生物活性的机制 结构是分子生物科学的主要未解决的问题之一， 对于合理的蛋白质设计，蛋白质 结构预测和折叠相关疾病状态。长期 该提案的目的是表征分子动力学， 蛋白质折叠早期事件的机制。全新 折叠的快速引发和表征方法 需要反应来实现该目标。这样一个项目的可行性 方法已经建立在实验中使用激光诱导 温度跃变和时间分辨红外光谱。因此，对于 第一次，有可能启动和跟踪蛋白质的动力学 以50 ps的时间尺度折叠或展开，或快10（7）倍 比常规的（快速混合）动力学研究。 这项工作的组织问题是什么是动态和 在分离的肽中和 蛋白质，以及二级结构形成的作用是什么， 引导折叠过程并稳定早期中间体。 具体来说，快速启动蛋白质折叠的新方法将 开发，使用激光诱导，脉冲宏观扰动， 时间分辨红外光谱法将被用作 蛋白质折叠中间体动力学的结构特异性探针。 肽和蛋白质的酰胺振动，以及同位素 标记将用于鉴定静态中的特定二级结构。 和时间分辨红外光谱。问题的层次将是 研究了从一系列从头螺旋肽开始， 研究螺旋形成的动力学和机理。 率 螺旋形核和传播，以及短程的作用， 将探索相互作用（静电、盐桥、封端）。 第二，快速碎片折叠的动力学和机制 肌红蛋白的折叠核心将被探索。短距离的影响 相互作用再次成为焦点，特别是反向的形成 转角和螺旋，以及它们在折叠反应中的相对作用 小肽最后，整个蛋白质（脱辅基肌红蛋白）将 一旦调查了第二代的动力学和红外特征， 结构要素的建立，以探讨相对的作用， 二级结构形成与长程相互作用（疏水的 在控制折叠反应的过程中。

The mechanism by which a protein folds to its native, biologically active structure is one of the major unsolved problems of molecular bioscience, with important practical consequences for rational protein design, protein structure prediction and folding related disease states. The long term objective of this proposal is to characterize the molecular dynamics and mechanisms of early events in protein folding. Fundamentally new approaches to the rapid initiation and characterization of folding reactions are required to fulfill this objective. The viability of such an approach has been established in experiments using a laser-induced temperature jump and time-resolved infrared spectroscopy. Thus, for the first time, it is possible to initiate and follow the kinetics of protein folding or unfolding on a time scale of 50 ps, or some 10(7) times faster than conventional (rapid mixing) kinetic studies. The organizing question of this work is what are the dynamics and mechanisms of secondary structure formation in an isolated peptide and in a protein, and what is the role of secondary structure formation in guiding the folding process and stabilizing early intermediates. Specifically, new methods for the rapid initiation of protein folding will be developed, using a laser-induced, impulsive macroscopic perturbation of temperature or pH. Time-resolved infrared spectroscopy will be used as a structure specific probe of the dynamics of protein folding intermediates. The amide vibrations of peptides and proteins, together with isotopic labeling will be used to identify specific secondary structures in static and time-resolved infrared spectra. A hierarchy of problems will be investigated. Starting with a series of de novo helical peptides, the dynamics and mechanism of helix formation will be investigated. The rate of helix nucleation and propagation, and the role of short range interactions (electrostatic, salt bridges, end capping) will be explored. Second, the dynamics and mechanism of folding of fragments of the fast folding core of myoglobin will be explored. The influence of short range interactions is again the focus, particularly the formation of reverse turns and helices, and their relative roles in the folding reactions of small peptides. Finally, the whole protein (apomyoglobin) will be investigated once the dynamics and infrared signatures of secondary structural elements are established, to probe the relative role of secondary structure formation versus long range interactions (hydrophobic collapse) in controlling the course of a folding reaction.