Probing Downhill Folding using Microfluidics and Single-molecule Fluorescence

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In this proposal the PIs will study how proteins fold in an active 3-dimensional structure. A fundamental prediction following from the landscape theory of protein folding is that protein folding can occur rapidly downhill on a multidimensional folding funnel. In this project the PIs will use a combination of single molecule fluorescence (SMF) and microfluidic methods to more directly explore downhill folding. The project will focus on the following specific goals. 1) Measure folding/unfolding rates of multiple putative downhill folding proteins using an ultrafast microfluidic mixer and ensemble fluorescence. 2) Directly probe the downhill character of folding for single protein molecules. Microfluidic mixing will trigger the folding/unfolding, and SMF will be used to follow the distribution of populations and dynamics during the transition. Non-equilibrium SMF/microfluidics methods, specifically with respect to time resolution and triggering will be improved for these experiments. 3) In order to further improve the time-resolution of the triggering, the PIs will also explore methods to photo-initiate folding of single protein molecules trapped in a new microfluidic device. The main intellectual merit of the proposed activity is to provide the first direct observations of this so-far elusive process, and provide a substantial test of the landscape theory of protein folding. These experiments will also be used to better examine many so-far hidden details of folding energy landscapes. The combined microfluidics/SMF methods developed during the project will be useful for a wide range of single-molecule investigations where the fast non-equilibrium kinetics needs to be resolved, and thus will be of general utility to the scientific community. The tools and general insights from this project will be disseminated widely. Undergraduate and post-doctoral students will receive training in evolving state-of-the-art technologies during the course of this interdisciplinary project involving two institutions and components from physics, biology and chemistry. Results from the project will also be incorporated into classes.