Quantifying the kinetics of conformational dynamics is key to understanding cell signaling networks. Traditional biochemical assays using recombinant systems report the rates of protein activities in an artificial aqueous environment, but in cells proteins are typically subject to a range of regulators operating on different aspects of conformational dynamics. These different regulatory mechanisms can be spatially localized, so they require tools to visualize rates of conformational change in living cells. Emergent live cell labeling systems such as SNAP and Halo tag are becoming increasingly valuable thanks to improvements in membrane-permeable dyes and could enable the use of environment-sensing dyes for single molecule quantitation of conformational change. Here, we explore the use of this strategy to study single-molecule GTPase conformational changes. The solvent-sensitive fluorescent dye Nile Red was attached to Cdc42 and Rac1 using a SNAP tag embedded in a linker connecting the GTPase to a peptide that binds only to the GTPase's active conformation. The probe was used to quantify local kinetics of membrane translocation and GTPase activation/inactivation. The subcellular distribution of rates indicates differential involvement of activation, deactivation, and membrane translocation throughout the cell.
Xu et al. (Sun,) studied this question.