Redox-Triggered Protein Folding Probed by Fluorescence Decay Kinetics

在天然状态下，蛋白质采用离散的构象或折叠，这被认为是组成多肽的最稳定的结构。某些化学物质或升高的温度可以破坏蛋白质的天然折叠，产生具有端到端距离广泛分布和大量构象的未折叠多肽的异质集合。蛋白质折叠研究的一个持续的挑战是理解这种异质集合如何演变成整齐折叠的蛋白质集合。开发合适的折叠探针是至关重要的。一个有效的折叠探针必须不仅报告未展开、中间和折叠形式的总体，而且还应该提供这些实体的结构同质性的信息。荧光蛋白折叠探针可以通过测量能量传递动力学来提供多肽集合的种群和异质性信息。在这个研究项目中，荧光衰减动力学将在蛋白质折叠反应中进行测量，以表征未折叠的集合到折叠蛋白质的演变。目标是开发和利用评价蛋白质折叠从触发到形成的整个过程中多肽结构和异质性的材料和方法。蛋白质折叠的复杂过程涉及从皮秒到分钟不等的时间尺度上的动力学。阐明折叠中的关键事件和确定任何部分折叠的中间体是理论和实验工作的主要目标。可以设想两种极限折叠机构。由于未折叠蛋白质的构象异质性，每个多肽可能遵循不同的折叠结构路径。在这种平行折叠极限下，转化动力学将是不均匀的。然而，如果只有少数可行的路径通向折叠结构，那么折叠动力学将会更简单，甚至可能类似于双态转换。区分这些限制机制的方法将在本研究计划中发展。从长远来看，这些研究将在蛋白质折叠转化的每个阶段提供多肽结构的观点。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学项目以及数学和物理科学理事会化学部门的无机、生物无机和有机金属化学项目支持。

MCB 9974477WinklerIn its native state, a protein adopts a discrete conformation, or fold, that is believed to represent the most stable structure for the constituent polypeptide. Certain chemicals or elevated temperatures can disrupt the native fold of a protein, producing a heterogeneous ensemble of unfolded polypeptides with a broad distribution of end-to-end distances, and a large number of conformations. A continuing challenge in protein-folding research is understanding how such a heterogeneous ensemble evolves into a collection of neatly folded proteins. The development of suitable probes of the folding process is of paramount importance. An effective folding probe must report not just on the populations of unfolded, intermediate, and folded forms, but also should provide information about the structural homogeneity of these entities. Fluorescent protein-folding probes can be engineered to provide both population and heterogeneity information for a polypeptide ensemble through measurements of energy-transfer kinetics. In this research program, fluorescence decay kinetics will be measured during protein-folding reactions in order to characterize the evolution of the unfolded ensemble into the folded protein. The goal is the development and utilization of materials and methods for evaluation of polypeptide structure and heterogeneity during the entire course of protein folding from triggering to formation of folded protein.The complex process of protein folding involves dynamics on time scales that range from picoseconds to minutes. Elucidating the key events in folding and identifying any partially folded intermediates are major goals of theoretical and experimental work. Two limiting folding mechanisms can be envisioned. Owing to the conformational heterogeneity of unfolded proteins, each polypeptide might follow a different path to the folded structure. In this parallel-folding limit, the kinetics of the transformation will be heterogeneous. If, however, only a small number of viable paths lead to the folded structure, then the folding kinetics will be simpler, perhaps even resembling those of a two-state transformation. Methods for distinguishing between these limiting mechanisms will be developed in this research program. In the long term, these studies will provide a view of polypeptide structure at each stage of the protein-folding transformation. This project is supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Inorganic, Bioinorganic and Organometallic Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.