Combined Single Molecule Force and Fluorescence Spectroscopy of the Unfolding and Refolding of Green Fluorescent Protein

Abstract

We have used optical tweezers to study the free energy surface for unfolding-refolding of the green fluorescent protein (EGFP) and simultaneously monitored the loss and recovery of its fluorescence. This conformational landscape shows unfolding intermediates, molten globe refolding intermediates, as well as misfolded states. As force is used to drive transitions between conformational substates, single molecule fluorescence is probed. In its native state, the emission of EGFP is punctuated by transient dark states (blinking); this emission is lost and regained as the protein is unfolded and subsequently refolded. These results provide a full understanding of the unfolding-refolding energy landscape of EGFP and how the conformational state affects the environment of the fluorophore, which reconciles three classes of previous experiments: (1) bulk unfolding/refolding with fluorescence (2) single molecule force-unfolding and (3) single molecule fluorescence intensity fluctuations. This investigation is relevant to efforts at developing EGFP as a genetically encoded force sensor, as well as its use in single molecule imaging and fluorescence recovery after photobleaching experiments.