Single-molecule investigation of ligand-activation mechanisms for membrane proteins in cell-derived nanovesicles

Molecular recognition of ligands for specific binding sites in membrane proteins is a cornerstone of biological signaling processes. Despite structural and functional evidence often painting a relatively clear picture of the binding sites, the mechanism by which ligand binding to multiple active sites confers a change in protein activity is in many cases still poorly understood. This is due in part to the fact that individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we describe an approach for resolving individual binding events of fluorescently labeled ligands at single membrane proteins in cell-derived nanovesicles. The approach involves simple transient transfection, does not use detergent solubilization so that proteins are maintained throughout in their native lipid bilayer, and provides solution access to either intracellular or extracellular binding domains. Furthermore, we describe a fully automated approach to idealization of fluorescence time series describing the binding dynamics at many molecules imaged simultaneously. As an exemplar, we apply these approaches to cyclic nucleotide binding to TAX-4 ion channels critical for sensory transduction and discuss mechanistic insights from kinetic modeling of their binding dynamics. This approach is broadly applicable to studies of binding dynamics for membrane proteins with extracellular or intracellular domains in native cell membrane.