Spectroscopic Study of Protein Folding Dynamics

描述（由申请人提供）：蛋白质通过其线性多肽链的特异性折叠获得其独特功能。错误折叠导致许多疾病，如囊性纤维化和各种神经退行性疾病。尽管人们对蛋白质的二级和三级结构的形成进行了大量的研究，并且研究蛋白质折叠的实验和理论技术也在不断完善，但对蛋白质折叠的定量和预测性理解仍然是不可能的。仍然有许多基本的问题，如：特异性和非特异性相互作用如何决定折叠途径，能量景观的粗糙度，热和动力学可及的构象亚态？特定的构象和折叠事件发生的时间范围是多少？解决这些问题提出了需要进一步的研究与静态和时间分辨光谱技术，可以提供必要的时间分辨率和结构灵敏度。因此，拟议的研究的主要目标是开发新的光谱方法和新的构象探针，可用于产生详细的结构解释的稳定和瞬态折叠物种及其动态在感兴趣的时间范围内。计划进行一系列详细的实验，以获得对蛋白质折叠问题各个方面的理解的新见解。技术目标是：（a）开发毫秒停流红外光谱仪;（B）进一步开发用于蛋白质折叠和结合研究的基于非天然氨基酸的新光谱方法;（c）研究蛋白质折叠中隐藏的中间体;（d）研究蛋白质折叠动力学的演变;（e）研究单个蛋白质分子的构象和折叠动力学。实现这些特定的目标，不仅会导致新的实验方法，蛋白质折叠和结合的研究，但也有新的见解，许多基本问题的理解蛋白质折叠，从而作出实际和直接的贡献领域。

DESCRIPTION (provided by applicant): Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done on the investigation of how the secondary and tertiary structures of proteins are formed and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions such as: How do specific and nonspecific interactions determine the folding pathways, the roughness of the energy landscape, the thermally and kinetically accessible conformation substates? On what range of timescales do particular conformational and folding events occur? Addressing these questions presents the need for further studies with static and time-resolved spectroscopic techniques that can provide the necessary time resolutions and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformational probes that can be used to generate detailed structure interpretations of the stable and transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain new insight into the understanding of various aspects of the protein folding problem. The technical goals are to: (a) develop a millisecond stopped-flow infrared spectrometer; (b) further develop new spectroscopic methods based on unnatural amino acids for protein folding and binding studies; (c) study the hidden intermediate in protein folding; (d) study the evolution of protein folding kinetics; (e) study the conformational and folding dynamics of single protein molecules. Achieving these specific aims should result in not only new experimental methods for protein folding and binding studies but also new insights into the understanding of many fundamental issues in protein folding, thus making practical and direct contributions to the field.