Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long seque...