Figure 1.
Scheme shows the substrate induced dimerization of hGBP1 and hydrolysis of GTP.
After the first phosphate cleavage, the GDP-bound enzyme dimer can undergo the second phosphate cleavage leading to the formation of GMP or irreversibly dissociates to give free GDP.
Figure 2.
Temperature dependent GTPase assays of wild type hGBP1.
The experiments were carried out by mixing a trace amount of radiolabeled (α-32P) GTP and 50 μM of unlabelled GTP.
Figure 3.
Logarithmic plot of kcat for GMP and GDP formation versus 1/T shows the nonlinear trend.
The temperature dependent GTPase assays were carried out as described in the material and methods. The solid lines drawn through the experimental data points show the trend of the plot.
Figure 4.
Energy diagram of GDP formation from GDP-bound enzyme dimer.
Schematic representation of the energy diagram of (hGBP1.GDP)2 dimer to (hGBP1.GDP) monomer and finally free GDP and hGBP1 during GDP formation from GDP-bound enzyme dimer. At high and low temperature, the activation energies were shown to be -ve and zero, respectively. For clarity, other parts of the reaction are not shown.
Figure 5.
GTPase assays of wild type hGBP1 at various concentrations of external GDP at 37 °C.
The concentration of unlabelled GTP was kept approximately 35 fold higher than the Kd of GppNHp for wild type hGBP1 (Kd for GppNHp to whGBP1 ~ 1.5 μM).
Figure 6.
Substrate induced dimerization of hGBP1 and the hydrolysis of GTP.
After the first phosphate cleavage, the GDP-bound enzyme dimer undergoes the second phosphate cleavage leading to the formation of GMP or reversibly dissociates into GDP-bound enzyme monomer, which further reversibly dissociates into free GDP and enzyme.
Figure 7.
Urea- induced denaturation studies of wild type hGBP1 in the absence or presence of various nucleotides as indicated in each panel.
The concentrations of the protein, GppNHp, GDP and GMP were kept 0.5, 100, 500 and 500 μM respectively. The mixture of wild type protein and the nucleotide was prepared in a reaction buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl and 5 mM MgCl2 and kept for an hour at 37 °C. Then the reaction mixture was incubated with urea at 37 °C. The fluorescence intensity at the emission maxima was plotted against concentration of urea. The solid lines drawn through the experimental data points show the fitted curves using Eq 1. The experiments were carried in triplicate.
Table 1.
Thermodynamic data extracted from the urea-induced denaturation studies, as described in materials and methods.
Figure 8.
▵Hm versus Tm plot for wild type hGBP1 in the absence or presence of various nucleotides.
Plots of ▵Hm vs Tm are shown for wild type hGBP1 in the absence or presence of GppNHp, GDP and GMP. The concentration of free protein and nucleotides were kept identical to urea-induced denaturation study. Heat-induced denaturation studies of the protein were carried out by measuring tryptophan fluorescence intensity from 20 to 85 °C with varying concentrations of urea (0-0.8 M). The data were fitted to Eq. 2 to obtain ▵Hm and Tm at each concentration of urea. The slope of the solid line drawn through the data points was used for the estimation of ▵Cp. The inset represents heat-induced denaturation curve in the absence of urea and the solid line drawn through the data points shows the fitted curve according to Eq. 2. The experiments were carried in triplicate.
Table 2.
Thermodynamic data extracted from the heat-induced denaturation studies, as described in materials and methods.
Table 3.
Steady-state kinetic parameters of the wild type and mutant hGBP1 proteins.
Figure 9.
▵GD of wild type and mutant hGBP1 in the absence and presence of GppNHp.
Heat-induced denaturation studies of wild type and mutant proteins were carried out with or without GppNHp. The experimental data were fitted using Eq. 2 to obtain ▵Hm and Tm. ▵Cp of the mutant proteins was assumed to be same as in wild type. With these values, the ▵GD at 37 °C was calculated using Eq. 3.