Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue

The millisecond timescale dynamics of activated Ras transiently sample a low‐populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small‐molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non‐hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non‐hydrolyzable analogues GppNHp and GppCH2p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors. Slowly does it: The correlated internal motions of GTPγS‐bound Ras have been quantified using relaxation dispersion NMR spectroscopy with site‐specific details, demonstrating its accurate preservation of the physiological dynamics network critical for the binding of allosteric ligands.