GTPase Assay.

GTPase assays were performed in 20 mM HEPES-NaOH, pH 7.5, 150 mM KCl, 4 mM MgCl2 containing 5 μM of the MM construct, and 1 mM GTP. The reaction was carried out at 37 °C. Upon starting the reaction, 2-μL reaction aliquots were withdrawn at different time points and mixed with 2 μL of 1 M HCl to stop the reaction and then diluted with 26 μL of HPLC buffer (100 mM potassium phosphate buffer, pH 6.5, 10 mM tetrabutylammonium bromide, 7.5% acetonitrile). Reaction products were quantified on an HPLC system from Agilent equipped with a Hypersil ODS-2 C18 column (250 × 4 mm). Each time-point measurement (five time points) was done in triplicate with the same sample. The specific hydrolysis rate for each protein concentration was computed by linear regression with error given by the standard deviation of the linear fit. To investigate the effect of Zn2+ on artificial dimer activity, 2.5, 10, 40, 80, 160, and 400 μM ZnSO4 were added to the standard reaction. For testing concentration-dependent GTPase activity, reactions were performed in standard buffer at RT with 5, 15, 30, and 50 μM enzyme. The competition GTPase assay was performed by adding 0.21 or 0.85 mM of GDP to the reaction. See SI Appendix, section S1.A.2 for details of the kinetic modeling of the competition assay.