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定折叠获得其独特的功能。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。尽管人们已经对蛋白质的二级和三级结构的形成进行了大量的研究，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无法实现。仍然有许多基本问题，如：特异性和非特异性相互作用如何决定折叠路径，能量景观的粗糙度，热和动力学可达的构象亚态？特定的构象和折叠事件在什么时间尺度范围内发生？为了解决这些问题，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灵敏度。因此，提出的研究的主要目标是开发新的光谱方法和新的构象探针，可用于生成稳定和瞬态折叠物种及其在感兴趣的时间范围内的动力学的详细结构解释。一组详细的实验计划，以获得新的见解，了解蛋白质折叠问题的各个方面。技术目标是：(a)研制一种毫秒止流红外光谱仪；(b)进一步发展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)研究蛋白质折叠中的隐藏中间体；(d)研究蛋白质折叠动力学的演化；(e)研究单个蛋白质分子的构象和折叠动力学。实现这些特定目标不仅会导致蛋白质折叠和结合研究的新实验方法，而且会对蛋白质折叠的许多基本问题的理解产生新的见解，从而对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.