The Rac1 protein (human Rac1, residues 1 to 184) was expressed in Escherichia coli cells as a glutathione-S-transferase (GST) fusion protein, as described previously (5, 7). To suppress the self-association into a dimer at concentrations more than 102 μM, we used an R66E mutant in all of the NMR experiments (7). The Rac1 mutants were constructed with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). E. coli BL21-CodonPlus(DE3)-RP cells (Agilent Technologies), transformed with the pGEX-6P-1 plasmid encoding Rac1, were grown at 37°C in LB media for preparing nonlabeled samples or in deuterated M9 media for preparing isotopically labeled NMR samples. When 15N labeling was required, 15NH4Cl was used as the sole nitrogen source. For the selective 13CH3 labeling of methyl groups, [3-13C, 2-2H]-l-alanine (200 mg/liter, for Alaβ), [methyl-13C, 3,3-2H2]-α-ketobutyric acid [50 mg/liter, for Ileδ1; Cambridge Isotope Laboratories (CIL)], [3-methyl-13C, 3,4,4,4-2H4]-α-ketoisovaleric acid (80 mg/liter, for Leuδ1, Leuδ2, Valγ1, and Valγ2; CIL), [methyl-13C]-l-methionine (100 mg/liter, for Metε; CIL), and [2,2,3,3-2H4]-succinic acid (2.5 g/liter; CIL) were added 1 hour before the induction. The production of the Rac1 protein was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside at 20°C for 16 hours. The protein was purified to homogeneity by chromatography on Glutathione Sepharose 4B resin (GE). After cleavage of the GST-tag with PreScission protease (GE), the protein was further purified by size exclusion chromatography using the HiLoad 26/60 Superdex 75 pg (GE) in buffer containing 20 mM tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol (DTT). To observe the amide signals, the purified protein was incubated at 37°C for 36 hours in MgCl2-free buffer to exchange the amide hydrogen atom from 2H to 1H. NMR samples were prepared by changing the buffer to 20 mM Hepes-NaOH (pH 7.0), 0.5 mM GDP, 5 mM MgCl2, and 5 mM DTT by sequential dilution and concentration with an Amicon Ultra Centrifugal Filter Unit, NMWL (nominal molecular weight limit) 10 kDa (Merck Millipore).

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