The Effect of Phosphoserine-Containing Membranes on Electrostatic Fields at the Protein-Protein Interface Measured through Vibrational Stark Effect Spectroscopy.

In the cell, Ras GTPases function as membrane-bound molecular switches for a variety of cell signaling pathways. Ras isoforms have long been of interest because of the connection between amino acid mutations and tumorigenesis. Much research focused on Ras has used truncated, solubilized constructs, which exclude the membrane-binding domain and therefore ignore the effects of membrane binding on Ras function. Since the membrane is a highly charged surface, it could have a significant impact on the electrostatic environment at or near the protein-protein interface. Here, we use a thiocyanate probe chemically inserted into the Ras-binding domain of RalGDS to investigate the effect of membrane binding at the Ras active site. Changes in the electric field caused by the membrane were measured by the probe as vibrational energy shifts in the infrared (IR) spectrum. For a selection of mutants which caused large shifts at this interface on the soluble H-Ras construct, binding to a 30% phosphatidylserine (PS)/70% phosphatidylcholine (PC) nanodisc caused reduced shifts compared to the solubilized counterparts. Additionally, the vibrational probe bonded to the wildtype (WT) Ras construct demonstrated a shift of 0.7 cm^-1 as a PC nanodisc was doped from 0% to 30% PS, but mutations introduced to the Ras active site caused the probe to show no shift across these PS concentrations. These results indicate that the local membrane environment has an effect on the electrostatics at the Ras active site and needs to be considered when investigating the effect of oncogenic mutations on Ras function.