Ras, a small GTPase, is central to the regulation of diverse cellular processes including transcription, cell cycle progression, growth, migration, cytoskeletal reorganization, apoptosis, cell survival, and senescence. Ras activation is mediated by GTP binding, whereas its inactivation occurs via GDP binding, which is tightly controlled by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). GAPs accelerate GTP hydrolysis, playing a crucial role in modulating Ras signaling to prevent excessive or prolonged activation. Here, we investigated monofunctional azobenzene derivatives as photochromic modulators to control the function of Ras in a light-dependent and reversible manner. Three thiol-reactive azobenzene derivatives with distinct electrostatic properties were synthesized and incorporated into GAP functional sites to modulate Ras activity. GAP mutants containing a single cysteine residue at the functional site were generated using an Escherichia coli expression system. Our results showed that modifications near the GAP "arginine finger," a critical region for stabilizing the GTP hydrolysis transition state of Ras, induced significant light-dependent changes in GTPase activity. We achieved photoreversible control of the interaction between Ras and its effector Raf using these azobenzene derivatives. These findings suggest that Ras function can be precisely modulated using photochromic molecules, providing a novel light-based approach for controlling Ras activity.
