Purification and Characterization of RHO GTPases

All proteins were produced using Escherichia coli and baculovirus-insect cell expression system as described [34 (link)]. Glutathione S-transferase (GST) fusion proteins were isolated by affinity chromatography on a glutathione Sepharose column in the first step and purified by size exclusion chromatography after proteolytic cleavage of GST in the second step [86 (link)]. His-tagged proteins were isolated from SF9 insect cells, using affinity chromatography on Ni-NTA columns. The quality of the proteins was analyzed by 12% SDS-PAGE. Protein concentrations were determined using Bradford reagent (Coomassie dye reagent; Sigma, (Steinheim, Germany)), and the GDP concentration in the case of purified RHO GTPases was determined using HPLC [87 ]. Nucleotide-free RHO proteins were prepared using alkaline phosphatase (Sigma Aldrich, Deisenhofen, Germany)and phosphodiesterase (Sigma Aldrich, Deisenhofen, Germany) at 4 °C as previously described [87 ]. RHO GTPases were loaded with 2-deoxy-3-O-N-methylanthraniloyl GDP (mdGDP); the fluorescent reporter group methyl-anthraniloyl (m) was attached to the 3′-OH group, and can, due to the lack 2′-OH, not isomerize between 2′- and 3-OH groups as compared to mGDP [88 (link)].