Redox-Triggered Protein Folding Probed by Fluorescence Decay Kinetics

MCB 9974477温克尔在其天然状态下，蛋白质采用离散的构象或折叠，被认为是组成多肽的最稳定的结构。某些化学物质或高温会破坏蛋白质的天然折叠，产生端到端距离分布广泛的未折叠多肽和大量构象。蛋白质折叠研究中的一个持续挑战是理解这样一个异质集合是如何进化成一组整齐折叠的蛋白质的。开发适合折叠过程的探头是至关重要的。一个有效的折叠探针不仅必须报告未折叠、中间和折叠形式的种群，而且还应该提供关于这些实体结构同质性的信息。可以设计荧光蛋白质折叠探针，通过测量能量转移动力学来为多肽集合提供种群和异质性信息。在这个研究项目中，将测量蛋白质折叠反应过程中的荧光衰减动力学，以表征未折叠的系综演变为折叠的蛋白质。其目标是开发和利用材料和方法来评价蛋白质折叠从触发到折叠蛋白形成的整个过程中的多肽结构和异质性。蛋白质折叠的复杂过程涉及从皮秒到几分钟的时间尺度上的动力学。阐明折叠过程中的关键事件和识别任何部分折叠的中间体是理论和实验工作的主要目标。可以设想两种限制折叠机构。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路径到达折叠结构。在这种平行折叠的极限下，转变的动力学将是不均匀的。然而，如果只有一小部分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。在这项研究计划中，将开发区分这些限制机制的方法。从长远来看，这些研究将提供蛋白质折叠转化每个阶段的多肽结构的观点。该项目得到了生物科学局分子和细胞生物科学部的分子生物物理学计划和数学和物理科学局化学部的无机、生物无机和有机金属化学计划的支持。

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.