The unfolding rate of Rac1 was measured by monitoring the reduction in the tryptophan fluorescence caused by protein unfolding (14). The purified Rac1 protein (1 μM) was prepared in buffer containing 50 mM tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 5 μM GDP, and 1 mM DTT. Then, the protein solution was rapidly mixed with an equivalent volume of buffer containing various concentrations of guanidine hydrochloride, and the time-dependent changes in the fluorescence were monitored. The unfolding rate under native conditions was determined by linear extrapolation to 0 M guanidine hydrochloride. The excitation and emission wavelengths were set to 281 and 340 nm, respectively, with slit widths of 5 nm. The measurements were performed at 25°C. The difference in the activation free energy was calculated according to ΔΔGunfold = –RTln(kunfold,WT/ kunfold,N92I), in which R denotes the gas constant and T represents the absolute temperature.

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