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。特异性和非特异性相互作用如何决定蛋白质折叠途径、天然和非天然结构、热动力学可及的构象亚态，以及特定折叠事件在什么时间尺度范围内发生，仍然存在许多基本问题。为了解决这些问题，需要进一步研究时间分辨光谱技术，以提供必要的时间分辨和结构灵敏度。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，可用于生成瞬态折叠物种的详细结构解释及其在感兴趣的时间范围内的动力学。我们计划进行一系列详细的实验，以深入了解折叠问题的各个方面，包括螺旋-线圈过渡、早期折叠事件和中间产物，以及β -薄片形成的机制。技术目标是：(a)研制一种纳秒温度跳变红外光谱仪，既能测量离散频率的瞬态动力学，又能测量离散反应时间的时间分辨光谱；(b)研制微秒级FTIR耦合连续流混合装置；(c)探索新的同位素编辑技术，以进行特定地点的构象研究；(d)将腈引入各种氨基酸中，作为蛋白质折叠、动力学和相互作用的红外探针；(e)研究螺旋-线圈过渡；(f)研究WW结构域β -片的形成及其与多肽的相互作用；(g)研究单个GFP分子的折叠和自发波动。

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 as to how specific and nonspecific interactions determine the protein folding pathways, the native and nonnative structures, and thermally and kinetically accessible conformation substates, and on what range of timescales do particular folding events occur. Addressing these questions presents the need for further studies with time-resolved spectroscopic techniques that can provide the necessary time resolution and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformation probes that can be used to generate detailed structure interpretations of the transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain detailed insight into the understanding of various aspects of the folding problem, including the helix-coil transition, early folding events and intermediates, and the mechanism of beta-sheet formation. The technical goals are: (a) to develop a nanosecond temperature jump infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times; (b) to develop a microsecond FTIR coupled continuous-flow mixing apparatus; (c) to explore novel isotope editing techniques to permit site specific conformation studies; (d) to introduce nitriles into various amino acids as infrared probes of protein folding, dynamics and interactions; (e) to study the helix-coil transition; (f) to study the beta-sheet formation in the WW domain and its interaction with peptides; (g) to study the folding and spontaneous fluctuation of single GFP molecules.