Single Molecule Study of the Processive Ras/SOS Interaction

Abstract

Ras is a membrane-anchored small GTPase protein that plays an important role in regulating essential cellular functions such as proliferation, differentiation, and apoptosis. Its deregulation is a hallmark of many cancers and developmental defects. Son of Sevenless (SOS) is a guanine nucleotide exchange factor (GEF) enzyme that activates Ras by catalyzing the exchange of GDP to the GTP in Ras.Previously, we have shown that in addition to the catalytic site, SOS has a catalytically inactive distal Ras-binding site, which allows SOS to localize and up-concentrate at Ras presenting membranes, dramatically increasing the Ras-GDP turnover rate. Together, the catalytic and allosteric sites form the catalytic core of SOS (SOScat). The existence of the extra binding site for Ras also raises the question of whether SOS is processive, capable of remaining surface bound via the distal binding site while catalyzing the nucleotide exchange of multiple Ras. In this study we employ various fluorescence-based methods such as Fluorescence Correlation Spectroscopy (FCS) and Total Internal Reflection Fluorescence (TIRF) microscopy on Ras functionalized supported lipid bilayers (SLBs) to demonstrate that SOScat is processive. Single molecule tracking of SOScat allows us to correlate the diffusion behavior between Ras and SOScat and further confirms the specific interaction.By confining individual SOScat enzymes to micron-scale two-dimensional Ras-functionalized SLB “reaction chambers” we can simultaneously monitor enzymatic activity from hundreds of single SOScat, probing the variability in catalytic rate and processivity within the enzyme ensemble. Our data indicates that SOScat has a broad range of processivities ranging from a few up to a thousand turnovers.