Redox-Triggered Protein Folding Probed by Fluorescence Decay Kinetics

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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.