Single-Molecule Protein Interactions and Unfolding Revealed by Plasmon-Enhanced Fluorescence

Single-molecule characterization of protein interaction kinetics can unravel crucial mechanisms that are averaged out with ensemble-average approaches. However, current approaches based on single-molecule fluorescence are limited in terms of signal brightness and time resolution. We introduce a novel platform to quantify protein-protein interactions at the single-molecule level using plasmon-enhanced fluorescence microscopy. We illustrate the power of this approach using PDZ protein that is conjugated to plasmonic particles using a novel DNA-mediated hybridization method that provides spatial and orientational control over the proteins' immobilization. Single-molecule kinetic studies uncover heterogeneities in the interaction where a subpopulation of events exhibits a distinct bound-state-lifetime not observed before. This new method also enables the study of urea-mediated unfolding and refolding using binding kinetics as readout. The bound-state lifetime was found to be independent of urea concentration, implying a simple two-state unfolding model. In addition, we find that the folding is entirely reversible for the immobilized PDZ, in contrast to solution-phase unfolding that results in aggregation. Altogether, our results present single-molecule plasmon-enhanced fluorescence as a new and powerful method to monitor transient protein-protein interactions and protein folding on short time scales.