Article Figures & Data
Figures
- Fig. 1.
Purification of His-tagged Era and specific binding to GTP and GDP. (A) Coomassie blue-stained gel containing uninduced (lane 1) and induced (lane 2) E. coli cells and the soluble (lane 3) and insoluble (lane 4) fractions obtained after lysis through a French pressure cell. (B) Silver-stained gel containing the Ni-NTA-purified Era (lane 1) and the fractions after further purification through anion exchange (lane 2) and gel filtration (lane 3). (C) Autoradiogram of Era-[γ-32P]GTP complexes separated by SDS-PAGE. For this assay, 250 pmol of Era and 2 pmol (10 μCi) of [γ-32P]GTP were prebound in binding buffer containing 5 mM MgCl2. Without UV cross-linking (lane 1), no Era-[γ-32P]GTP complexes are observed. UV cross-linking resulted in Era-[γ-32P]GTP complexes detected without competing nucleotide (lane 2) or in the presence of 800 pmol GTP, GDP, GMP, ATP, CTP, or UTP (lanes 3 to 8). Era-[γ-32P]GTP binding is inhibited by excess GTP and GDP (lanes 3 and 4, respectively).
- Fig. 2.
Excitation spectra for mGDP and mGTP alone or bound to Era. Excitation spectra from 310 to 410 nm were recorded at an emission wavelength of 446 nm in the presence of 5 mm MgCl2. The light lines (superimposed) represent the fluorescence signal from 0.1 μM mGDP or mGTP in the absence of Era; the fluorescence intensity of the mant moiety is identical when coupled to GDP or GTP. Upon addition of Era (5 μM), the fluorescence intensities of both mGDP and mGTP increase. The fluorescence signal from Era-mGDP is shown by the heavy solid line, and the fluorescence signal from Era-mGTP is shown by the heavy dotted line.
- Fig. 3.
Association curves for binding of Era to mGDP and mGTP. Era (1 μM) and 0.5 to 20 μM mGDP (A) or mGTP (B) in binding buffer containing 5 mM MgCl2 were mixed in a stopped-flow apparatus, and the increase in fluorescence intensity was recorded over time. Data were fitted to a double exponential equation, and the rate constants and amplitude values were calculated. Curves shown represent the average of three separate data sets.
- Fig. 4.
Equilibrium binding constants for Era and mGDP or mGTP. The equilibrium binding constant, KD, was obtained for mGDP and mGTP by plotting total amplitudes from the association curves (Fig. 3) against final concentration of mant nucleotide and fitting to a one site binding equation; due to the lowKD value, the mGDP data were subsequently corrected for depletion of nucleotide due to binding. (A) Single set of data for mGDP KD; (B) average of three data sets for mGTP KD.
- Fig. 5.
Guanine nucleotide exchange by Era. Data from a typical mant nucleotide exchange assay are shown, in this case exchange of mGTP for GDP in the presence of 5 mM MgCl2. Era (3 μM) was prebound to 0.3 μM mGTP in binding buffer. This solution was mixed with a 45 μM solution of GDP in a stopped-flow apparatus, and the decrease in fluorescence intensity was recorded over time. Data (black solid line) were fitted to a single (black dotted line) or double (gray line) exponential equation. The exchange rates,koff, and amplitude values were calculated from the double exponential equation.
- Fig. 6.
Association of mant nucleotides with Era. Data from a typical mant nucleotide association assay, in this case 0.5 μM Era and 0.5 μM mGDP in the presence of 5 mM MgCl2. Era (1 μM) and 1 μM mGDP in binding buffer were mixed in a stopped-flow apparatus, and the increase in fluorescence intensity was recorded over time. Data (light line) were fitted to a double exponential equation (heavy line), and the association rate constants,kon, and amplitude values were calculated.
- Fig. 7.
Hydrolysis of mGTP by Era in the presence of 0.2 mM MgCl2. Data are from a typical hydrolysis assay in which 0.1 μM mGTP was prebound to 5 μM Era in binding buffer containing 0.2 mM MgCl2, and the decrease in fluorescence intensity due to hydrolysis of mGTP to mGDP was recorded over time (points). Data were fitted to a single exponential decay curve (solid line), and the single-turnover hydrolysis rate was calculated. (Inset) Excitation spectra (excitation, 310 to 410 nm; emission, 446 nm) demonstrating the difference in fluorescence intensity of Era-mGTP and Era-mGDP in the presence of 0.2 mM MgCl2. The light lines (superimposed) represent the fluorescence signal from 0.1 μM mGDP or mGTP in the absence of Era; the fluorescence intensity of the mant moiety is the same when coupled to GDP or GTP. Upon addition of Era (5 μM), the fluorescence intensities of both mGDP and mGTP increase. The fluorescence signal from Era-mGDP is shown by the heavy solid line, and the fluorescence signal from Era-mGTP is shown by the heavy dotted line.
Tables
- Table 1.
Dissociation rate constants for Era-mGDP and Era-mGTPa
Substrate Era-mGDP Era-mGTP koff(s−1) Contribution of kofffast (%) koff(s−1) Contribution of kofffast (%) Fast Slow Fast Slow MgCl2 5 mM 2.5 ± 0.2 0.27 ± 0.08 55 ± 12 8.8 ± 0.2 0.34 ± 0.01 68 ± 11 0.2 mM 3.7 ± 0.6 0.68 ± 0.05 32 ± 1 4.0 ± 1.1 0.60 ± 0.04 42 ± 3 1 mM EDTA 4.5 ± 0.9 0.74 ± 0.06 31 ± 2 14.8 ± 0.4 0.26 ± 0.03 75 ± 3 ↵a Error parameters are derived from the average of three separate data sets.
- Table 2.
Association rate constants for Era-mGDP and Era-mGTPa
Substrate Era-mGDP Era-mGTP kon (μM s−1) Contribution of kon fast (%) kon (μM s−1) Contribution of kon fast (%) Fast Slow Fast Slow MgCl2 5 mM 6.3 ± 0.6 1.6 ± 0.2 51 ± 6 11.0 ± 0.8 2.0 ± 0.3 68 ± 3 0.2 mM 15.2 ± 2.9 2.0 ± 0.1 24 ± 3 10.0 ± 0.5 1.1 ± 0.2 77 ± 2 1 mM EDTA 7.2 ± 0.7 1.6 ± 0.1 33 ± 3 9.0 ± 0.5 1.1 ± 0.3 86 ± 2 ↵a Error parameters are derived from the average of three separate data sets.
