Nanovesicle trapping for studying weak protein interactions by single-molecule FRET.

Protein-protein interactions are fundamental biological processes. While strong protein interactions are amenable to many characterization techniques including crystallography, weak protein interactions are challenging to study because of their dynamic nature. Single-molecule fluorescence resonance energy transfer (smFRET) can monitor dynamic protein interactions in real time, but are generally limited to strong interacting pairs because of the low concentrations needed for single-molecule detection. Here, we describe a nanovesicle trapping approach to enable smFRET study of weak protein interactions at high effective concentrations. We describe the experimental procedures, summarize the application in studying the weak interactions between intracellular copper transporters, and detail the single-molecule kinetic analysis of bimolecular interactions involving three states. Both the experimental approach and the theoretical analysis are generally applicable to studying many other biological processes at the single-molecule level.