EARLY EVENTS IN PROTEIN FOLDING

蛋白质折叠为天然的生物活性结构的机制是现代结构生物学尚未解决的主要问题之一，对合理的蛋白质设计、蛋白质结构预测和折叠相关疾病状态具有重要的实际意义。这项提议的中心目标是描述蛋白质折叠早期事件的分子动力学和机制。从根本上说，需要新的方法来快速启动和表征折叠反应，以实现这一目标。这种方法的可行性已经在使用激光诱导的温度跳跃和时间分辨红外光谱的实验中得到了证实。因此，第一次有可能在50ps的时间尺度上启动和跟踪蛋白质折叠或展开的动力学，或者说比传统的(快速混合)动力学研究快约107倍。这项工作的组织问题是，在分离的多肽和蛋白质中，二级结构形成的动力学和机制是什么，以及二级结构形成在指导折叠过程和稳定早期中间体方面起到什么作用。具体地说，将开发新的方法来快速启动蛋白质折叠，使用激光诱导的、脉冲的pH温度宏观扰动。时间分辨红外光谱将被用来作为蛋白质折叠中间体动力学的结构特异性探针。多肽和蛋白质的酰胺和侧链振动以及同位素标记将用于识别静态和时间分辨红外光谱中的特定结构。将对一系列问题进行调查。从一系列从头开始，我们将研究螺旋、转角和片状形成的动力学和机制。将探索成核和传播的速度，以及局部相互作用(静电、盐桥、末端封顶)的作用。其次，我们将探索无肌红蛋白快速折叠核心片段的折叠动力学和折叠机制。局部相互作用的影响再次成为焦点，特别是反转和螺旋的形成，以及它们在小肽折叠反应中的相对作用。最后，我们将研究两种蛋白质，无肌红蛋白(球状螺旋蛋白)和CSPA(快速折叠的β-Barrel)的折叠动力学。这项工作的一个关键焦点是确定局部和非局部(链内)相互作用在塑造能量格局中的作用，从而确定蛋白质折叠的动力学。

The mechanism by which a protein folds to its native, biologically active structure is one of the major unsolved problems of modern structural biology, with important practical consequences for rational protein design, protein structure prediction and folding related disease states. The central 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 107 times faster than conventional (rapid mixing) kinetics 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 of pH. Time-resolved infrared spectroscopy will be used as a structure specific probe of the dynamics of protein folding intermediates. The amide and sidechain vibrations of peptides and proteins, together with isotopic labeling will be used to identify specific structures in static and time-resolved infrared spectra. A hierarchy of problems will be investigated. Starting with a series of de novo peptides, the dynamics and mechanism of helix, turn and sheet formation will be investigated. The rate of nucleation and propagation, and the role of local interactions (electrostatic, salt bridges, end capping) will be explored. Second, the dynamics and mechanism of folding of fragments of the fast folding core of apomyoglobin will be explored. The influence of local 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 folding dynamics of two proteins, apomyoglobin (globular helical protein) and CspA (fast-folding beta-barrel) will be investigated. A key focus of this work is to establish the role of local and nonlocal (intrachain) interactions in shaping the energy landscape, hence the dynamics of protein folding.