Developing new single-molecule tools to study multi-protein complexes

Studying multi-protein complexes at the single-molecule level provides additional challenges as compared to single enzymes. The Kd’s that govern the association of the various protein components are often much higher than the concentrations permissible for single-molecule imaging. We aim to develop generalized approaches to studying fluorescently labeled proteins at physiological concentrations through the application of photoswitchable fluorophores and nanophotonics. Additionally, single-molecule assays capable of correlating structure and function are critical in describing the dynamics of multi-protein machines. In recent work, we have demonstrated a powerful new assay that combines nanomanipulation of DNA with observation of fluorescently labeled proteins to measure the activity and composition of the replisome, the multi-protein complex that carries out DNA replication (Loparo et al, PNAS 2011). This assay is broadly applicable to any number of multi-protein complexes acting on DNA and future efforts will build on this work by focusing on improving structural sensitivity and spatial resolution.