Inverse Temperature Jump System to Study Fast Protein Folding Kinetics

Abstract

A protein's conformation depends on the protein's chemical and physical environment, including the temperature, pH, and denaturant concentration. The changes in protein structure can be visualized by Forster resonance energy transfer (FRET) between donor and acceptor fluorescent dyes bound to separate residues on the protein. One method of studying protein folding kinetics is the temperature jump, where a protein sample is quickly heated by a laser pulse to trigger a change in the protein conformation. During and after the pulse, a fluorescence excitation laser illuminates the protein sample and the FRET signal is collected. Traditional temperature jump methods have very short heating times (∼1 ns), facilitating the observation of protein folding events triggered by heating. However, due to the relatively large heated volume (∼1 nL), the long cooling time (hundreds of μs) obscures cooling-driven protein folding events (tens of μs). In order to observe fast folding events, the protein sample is often placed in non-native conditions: its unfolded state is imposed by low temperatures, usually at a high denaturant concentration, so that the heating laser pulse will induce the protein to refold. We have designed a novel system, which we call the inverse temperature jump or iT-jump, that enables observations of fast folding transitions by cooling the protein sample on a timescale of <1 μs. The rapid cooling is made possible by placing the sample in a shallow microfluidic channel and tightly focusing the heating laser beam to a volume of ∼1 fL. The proposed system inverts the existing T-jump paradigm, as the heating pulse unfolds the protein and the fast cooling transition allows it to refold. As a demonstration of our system's capabilities, we have studied the folding behavior of BBL, a small, fast-folding protein, under various denaturant conditions.