A New Fluorescence Technique for Protein-Folding Studies

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ABSTRACT9876443Jay WinklerCalifomia Institute of TechnologyA New Fluorescence Technique for Protein-folding StudiesA heterogeneous ensemble of unfolded protein molecules should exhibit a broad, nearly Gaussian distribution of end-to-end distances, consistent with a large number of degenerate conformations and, hence, a high configurational entropy. Proteins in their native states, by contrast, have definite three-dimensional orientations of their peptide backbones and, to a certain extent, their side chains as well. The conformational freedom in folded proteins is far smaller than that of random polymers and, consequently, the configurational entropy is considerably lower than that of the unfolded ensemble. The energy landscape representing the conversion of a heterogeneous ensemble of unfolded proteins into a uniformly folded collection is expected to resemble a funnel. A continuing challenge in protein-folding research is understanding how a heterogeneous ensemble of unfolded polypeptides 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. The rate of dipole-dipole fluorescence energy transfer (FET) from an excited donor * D to an acceptor A varies as the inverse sixth power of the DA separation. If D and A are incorporated into a single protein molecule, measurements of *D fluorescence decay kinetics can be used to extract distributions of DA distances. Measurements of fluorescence decay kinetics during a protein-folding reaction can distinguish between a two-state folding mechanism and one in which the unfolded ensemble continuously evolves into the folded protein. The goal of this research program is the development of instrumentation and methods for evaluation of DA distance distributions during the entire course of protein folding from triggering to formation of folded protein.