The Tm values were obtained from the temperature-dependent changes in the molar ellipticity. The purified Rac1 protein (10 μM) was prepared in buffer containing 50 mM tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 10 μM GDP, and 1 mM DTT, and its molar ellipticity at 222 nm was monitored. The temperature was changed from 30° to 90°C in 1.5°C increments, with 30 s of equilibration time. The Tm values were obtained by fitting the temperature-dependent changes in molar ellipticity by using the Gibbs-Helmholtz equation (29).

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