Single Molecule Studies of Protein Folding Mechanisms

描述(申请人提供)：研究蛋白质折叠机制和蛋白质的部分折叠状态可以加深对许多人类疾病状态的理解。由于折叠过程往往非常复杂，传统的集成方法无法解决其动力学的几个关键特征。我们建议开发单分子荧光方法来研究多步蛋白质折叠反应中的详细构象分布和动力学。将开发监测蛋白质短距离变化的方法，并用于蛋白质的非扰动固定。还将进一步开发和优化单分子FRET和荧光寿命数据采集和分析方法。这些方法和相关的单分子方法将被应用于研究Barnase的多步折叠反应。使用单分子FRET和短程猝灭相结合的方法将获得关于不同折叠状态及其相互转化的结构信息。利用蛋白质工程，将检查突变对观察到的单分子分布和折叠景观的影响，并将澄清长期存在的问题，如状态之间的连通性和它们之间的跃迁的协同性。将研究分子拥挤和伴侣对这种折叠反应的影响，为蛋白质折叠的体内机制提供见解。最后，我们将研究Barnase折叠和与Barstar结合之间的相互作用。预计所开发的方法将在疾病相关蛋白的研究中有用。

DESCRIPTION (provided by applicant): Studies of protein-folding mechanisms and partially folded states of proteins can lead to a deeper understanding of many human disease states. Because the folding process is often very complex, traditional ensemble methods are unable to resolve several key features of its dynamics. We propose to develop single-molecule fluorescence methods for the study of detailed conformational distributions and dynamics during multi-step protein folding reactions. Methods to monitor short-range distance changes in proteins, and for the non-perturbative immobilization of proteins will be developed. Further development and optimization of single-molecule FRET and fluorescence lifetime data acquisition and analysis methods will also be carried out. These and related single molecule methods will be applied to study the multi-step folding reaction of barnase. Structural information about the different folding states and their interconversion will be obtained using a combination of single-molecule FRET and short-range quenching. Using protein engineering, the influence of mutations on the observed single-molecule distributions and folding landscapes will be examined, and long-standing issues such as connectivity between states and cooperativity of transitions between them will be clarified. The influence of molecular crowding and chaperones on this folding reaction will be studied, providing insights into in-vivo mechanisms of protein folding. Finally, the interplay between barnase folding and binding to barstar will be studied. It is anticipated that the developed methods will be useful in the study disease-related proteins.