Figure 4. FtsY binding to SecYEG monitored by fluorescence. (A) Equilibrium titrations of SecYEG-FtsY complex formation monitored by FRET. SecYEG embedded into nanodiscs was titrated with FtsY in the absence of guanine nucleotide (○) or in the presence of 0.5 mM each of GDP (□), GTP (⋄), or GMP-PNP (△). SecY was labeled with the donor fluorophore MDCC at position 111 and FtsY with the acceptor fluorophore BODIPY FL at position 196 [FtsY(Bpy)]. Donor fluorescence is plotted relative to the initial fluorescence measured before FtsY(Bpy) addition and set to 1.0. Plotted are mean values from two titrations; SEMs were ≤5%. Kd values were obtained by nonlinear fitting using equation 1 (see Materials and methods); errors are SEMs of the fits. (B) Equilibrium titrations of FtsY binding to SecYEG in nanodiscs (○) and to empty nanodiscs (●) monitored by the fluorescence increase of an NBD label attached to position 26 of FtsY, corrected for the linear signal increase measured upon titrating FtsY(NBD) into buffer (Materials and methods). Plotted are mean values from two titrations each; error bars represent SEM. Upon complex formation, the fluorescence signal of FtsY(NBD) increased 6–8-fold; to facilitate comparison, the fluorescence increase is plotted in normalized numbers. Kd values of 0.16 ± 0.02 µM (SecYEG in nanodiscs) and 1.2 ± 0.4 µM (empty nanodiscs) were determined by nonlinear fitting using equation 1 (Materials and methods); errors are SEMs of the fits. (C) Inhibition of FtsY binding to SecYEG in the presence of nonionic detergents. The fluorescence of SecYEG(MDCC) (0.05 µM) embedded in nanodiscs or solubilized by detergent, as indicated, was monitored on addition of FtsY(Bpy) (1 µM). The fluorescence signal measured after the addition of FtsY(Bpy) is plotted relative to the respective initial signal set to 1.0. The error bars indicate the SD (n = 3).
Figure 4.

FtsY binding to SecYEG monitored by fluorescence. (A) Equilibrium titrations of SecYEG-FtsY complex formation monitored by FRET. SecYEG embedded into nanodiscs was titrated with FtsY in the absence of guanine nucleotide (○) or in the presence of 0.5 mM each of GDP (□), GTP (⋄), or GMP-PNP (△). SecY was labeled with the donor fluorophore MDCC at position 111 and FtsY with the acceptor fluorophore BODIPY FL at position 196 [FtsY(Bpy)]. Donor fluorescence is plotted relative to the initial fluorescence measured before FtsY(Bpy) addition and set to 1.0. Plotted are mean values from two titrations; SEMs were ≤5%. Kd values were obtained by nonlinear fitting using equation 1 (see Materials and methods); errors are SEMs of the fits. (B) Equilibrium titrations of FtsY binding to SecYEG in nanodiscs (○) and to empty nanodiscs (●) monitored by the fluorescence increase of an NBD label attached to position 26 of FtsY, corrected for the linear signal increase measured upon titrating FtsY(NBD) into buffer (Materials and methods). Plotted are mean values from two titrations each; error bars represent SEM. Upon complex formation, the fluorescence signal of FtsY(NBD) increased 6–8-fold; to facilitate comparison, the fluorescence increase is plotted in normalized numbers. Kd values of 0.16 ± 0.02 µM (SecYEG in nanodiscs) and 1.2 ± 0.4 µM (empty nanodiscs) were determined by nonlinear fitting using equation 1 (Materials and methods); errors are SEMs of the fits. (C) Inhibition of FtsY binding to SecYEG in the presence of nonionic detergents. The fluorescence of SecYEG(MDCC) (0.05 µM) embedded in nanodiscs or solubilized by detergent, as indicated, was monitored on addition of FtsY(Bpy) (1 µM). The fluorescence signal measured after the addition of FtsY(Bpy) is plotted relative to the respective initial signal set to 1.0. The error bars indicate the SD (n = 3).

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