Abstract
In Ras activation, the Ras-specific nucleotide exchange factor Son of sevenless (SOS) plays critical roles in trnasducing receptor stimuli into Ras as well as shaping bimodal distribution of Ras activity. SOS consists of multiple modular domains that prevent the unchecked activation of Ras and also fully activate its own activity in response to receptor ligation. The molecular behavior of SOS is multilayered and controlled by complicated intra- and inter-molecular interactions in the context of membrane environments. Although great details of regulatory mechanisms of SOS have been revealed using purified, truncated SOS variants (C-terminal truncated SOS-HDPC, 1-1050) in vitro, the molecular kinetics of native full-length SOS is not yet well studied, mainly owing to difficulties in purifying full-length proteins. Here, we developed purification-free, cell-lysate based single molecule enzyamtic assays to sutdy molecular kinetics of full-length SOS on a supported lipid bilayer. We observed that allosterically activated full-length SOS exhibits stochastic fluctuation of catalytic activity and similar enzymatic rate distribution as observed in SOS-HDPC. In addition, C-terminal domain provides an additional layer of autoinhibition of SOS by reducing basal interactions with Ras. This mehtod could be generalizable to study enzymatic activity of native proteins on the membranes at the single molecule level.
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