Abstract
Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP‐to‐GTP exchange. This modifies the Ras surface (Ras~on~GTP) and enables effector binding, which then activates signal‐transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to “off” (Ras~off~GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras~off~‐to‐Ras~on~ transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time‐resolved FTIR (trFTIR) spectroscopy. In the transition from Ras~off~ to Ras~on~ a GTP‐bound intermediate is now identified, in which effector binding is still prevented (Ras~off~GTP). The loop movement from Ras~off~GTP to Ras~on~GTP was directly monitored by the CO vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s^−1^ at 260 K. A small molecule that shifted the equilibrium from the Ras~on~GTP state towards the Ras~off~GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.