Signaling networks maintain their spatiality by localization of their protein constituents to distinct regions of the membrane, and the different Ras guanine nucleotide binding proteins are a paradigm example of this. Despite being highly homologous, they exhibit isoform specific diversity in generating explicit signal outputs governed by, but not limited to, their hyper variable region responsible for targeting them to particular membrane microdomains (1). In addition, Ras proteins are known to sample multiple conformations which exhibit varying affinities towards their interaction partners. To fully explore the conformational space exhibited by Ras, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We applied pressure modulation in combination with FT-IR spectroscopy to reveal equilibria between spectroscopically resolved, otherwise low lying, substates of the lipidated signaling protein N-Ras in its different nucleotide binding states and in the absence and presence of a model biomembrane. Not only the nucleotide binding, but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G-domain involving α-helix-membrane interactions. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner which has consequences for the G-domain orientation of membrane-associated N-Ras which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide first insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea.Reference1. Weise K, Kapoor S, ... Waldmann H, Winter R (2011) J Am Chem Soc 133:880-887.Table of Contents