Spectroscopic Study of Protein Folding Dynamics

描述(申请人提供)：蛋白质通过其线性多肽链的特定折叠获得其独特的功能。错误折叠会导致许多疾病，如囊性纤维化和各种神经退行性疾病。尽管人们在研究蛋白质的二级和三级结构是如何形成的方面做了大量的工作，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，例如：特定的和非特定的相互作用如何决定折叠路径、能量景观的粗糙度、热和动力学上可访问的构象亚态？特定的构象和折叠事件在什么时间范围内发生？解决这些问题需要进一步研究静态和时间分辨光谱技术，以提供必要的时间分辨率和结构敏感性。因此，拟议研究的主要目标是开发新的光谱方法和新的构象探针，可用于对稳定和瞬时折叠物种及其在感兴趣的时间范围内的动力学进行详细的结构解释。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。其技术目标是：(A)开发毫秒停流红外光谱仪；(B)进一步开发基于非天然氨基酸的新的光谱方法，用于蛋白质折叠和结合研究；(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，不仅将为蛋白质折叠和结合研究带来新的实验方法，而且将为理解蛋白质折叠中的许多基本问题提供新的见解，从而为该领域做出实际和直接的贡献。

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.