Fig. 3

Biochemical properties of Obg_GC. a GTPase cycle. Obg GTPases oscillate between active (ON, GTP-bound) and inactive (OFF, GDP-bound) states. b Obg_GC binds mant-GTP and mant-GDP with guanine nucleotide-specific Mg²⁺ dependence. Binding of mant-GTP (red circles) and mant-GDP (blue squares) to N-His-Obg_GC was assessed in the presence of varying concentrations of Mg²⁺. The averages with SEM from three independent experiments are shown. c Increase in Relative Fluorescence Units (RFU) of mant-GTP (red bars) and mant-GDP (blue bars) upon addition of different Obg_GC variants: recombinant wild type Obg_GC with N- and C-terminal 6 × His tag (N-His-Obg_GC and C-His-Obg_GC, respectively), and N-His-Obg_GC with T192AT193A substitutions. The data shows averages with corresponding SEM of at least eight experiments performed on separate occasions. d Hydrolysis of mant-GTP by N-His-Obg_GC (green) and C-His-Obg_GC (red) was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes. Data from at least four experiments were fitted to a single exponential decay equation. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black