Figure 8: Biochemical properties of the Enterococcus hirae V1 complex.
From: Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor
(a–d) Isothermal titration calorimetry (ITC) analysis. Nucleotides (200 μM) were injected into 7 μM EhV1 at 25 °C. The integrated heat values from raw heats (inset) were plotted against the molar ratio of nucleotides to EhV1 after subtraction of the nucleotide dilution heat values from the corresponding heat values of the EhV1-nucleotide titration. (a,b) show the binding isotherm titrated to nucleotide-free EhV1 with AMP-PNP (a) and ADP (b). The solid line represents the best fit to a binding model including the two sets of sites model for AMP-PNP (a) and the three sets of sites model for ADP (b). (c) shows the binding isotherm titrated to ADP-bound EhV1 with AMP-PNP. (d) shows the binding isotherm titrated to AMP-PNP-bound EhV1 with ADP. (e) Tryptophan fluorescence changes of nucleotide-free EhV1 by addition of 500 nM (lane 1), 21 μM (lane 2) and 100 μM (lane 3) AMP-PNP and 500 nM (lane 4), 35 μM (lane 5) and 100 μM (lane 6) ADP. (f) Tryptophan fluorescence changes of 35 μM ADP-bound and 21 μM AMP-PNP-bound EhV1 from nucleotide-free EhV1 by addition of 2 mM AMP-PNP (lane 1) and 2 mM ADP (lane 2), respectively. The intensity was averaged between 330 and 340 nm. All data represent means±standard estimated errors (s.e.m.) of three independent experiments.
