Dissecting the Rate Theory for Protein Folding Dynamics via Advanced Single-Molecule Fluorescence Experiments

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Proteins are biological nanomachines that perform most cellular functions through their ability to self-assemble into specific structures and change shape on cue. The mechanisms that govern protein operation determine every aspect of molecular and cell biology and are also essential for developing tools to predict, engineer, and design biological function. The keys to these mechanisms are in the rates at which proteins fold and unfold. However, there still is not a unified rate treatment that can: 1) integrate analytical theory and molecular simulations; 2) rationalize the puzzlingly simple rate patterns observed in conventional experiments; and 3) be directly used to analyze modern single-molecule experiments. This project will demonstrate a novel approach to empirically dissect folding rates. It will also provide answers to many outstanding issues in the field, including how the rates depend on various physical factors and protein fold/structure, the connections between rate and mechanism, and how heterogeneous these mechanisms truly are. Capitalizing on the special characteristics of the University of California Merced, a Hispanic serving institution with over 60% of first-generation students, the project will help develop a robust pipeline for underrepresented minorities in the STEM workforce at all levels and produce graduates that can pursue cross-disciplinary STEM careers in academia, government laboratories or industry. The effective bridge between predictions of theory and simulations and the analysis of experiments in protein folding is a rate treatment that describes rates as diffusion on the free energy surface obtained by projecting the energy landscape into a few order parameters. To effectively test this treatment, experimentalists must resolve the distribution of microscopic behaviors that inform on the rate’s properties, a feat that has not been yet fully achieved. The goal of this project is to experimentally dissect the rates of protein folding-unfolding transitions. This will be done using an approach designed to track the stochastic changes that individual protein molecules undergo along their reactive transition paths, and hence resolve the key rate terms: barrier, diffusion coefficient, and curvature of wells and barrier top. The approach combines advanced single-molecule fluorescence experiments, maximum likelihood analysis of photon trajectories, and diffusive kinetic models. This approach will be applied to fast-folding proteins representatives of three basic archetypal folds. The experiments will directly test protein folding rate theory, connect with molecular simulations, and promote a paradigm shift in how experimental rates are interpreted.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.