Figure 6. SecYEG-FtsY complex formation in the presence of FtsQ-RNC, HemK-RNC, or SRP. (A) SecYEG-FtsY complex formation in the presence of FtsQ-RNC. MDCC-labeled SecYEG in nanodiscs was titrated with FtsY(Bpy) in the presence of increasing concentrations of FtsQ-RNC (µM): 0, ●; 0.005, ○; 0.01, ■; 0.02, □; 0.05, ▲; 0.1, △. For clarity, representative error bars (SEM; n = 2) are indicated only on the last titration point. Kd values of ∼0.18 µM were determined by nonlinear fitting using equation 1 (Materials and methods); at concentrations of FtsQ-RNC >0.02 µM, the fluorescence change was too small to allow for the estimation of reliable Kd values. (B) Effect of FtsQ-RNC binding on the SecYEG-FtsY complex. Kd values from A are plotted against the concentration of FtsQ-RNC (○; right Y-axis). Donor (MDCC) fluorescence measured at saturation with FtsY(Bpy) (●, left Y-axis) is plotted relative to the initial fluorescence of SecYEG(MDCC) measured before the addition of FtsY(Bpy); error bars represent SEM from (A). Nonlinear fitting to equation 2 (Materials and methods) yielded an apparent Kd = 8 ± 1 nM for the binding of FtsQ-RNC to the SecYEG-FtsY complex. (C) SecYEG-FtsY complex formation in the presence of HemK-RNC. Titrations were performed as in A in the presence of increasing concentrations of HemK-RNC (µM): 0, ●; 0.03, ○; 0.08, ■; 0.25, □. (D) No effect of HemK-RNC on SecYEG-FtsY complex formation. (E) SecYEG-FtsY complex formation in the presence of SRP. MDCC-labeled SecYEG was titrated with FtsY(Bpy) in the presence of increasing concentrations of SRP (µM): 0, ●; 0.05, ○; 0.1, ■; 0.2, □; 0.3, ▲; 0.5, △. Apparent Kd values of ∼0.2 µM (○, right Y-axis) were determined by nonlinear fitting as in A. (F) Effect of SRP on the SecYEG-FtsY complex. Kd values from C are plotted against the SRP concentration (○). Donor (MDCC) fluorescence measured at saturation (●, left Y-axis) is plotted relative to the initial fluorescence measured before the addition of FtsY(Bpy). Nonlinear fitting as in B yielded an apparent Kd of 40 ± 10 nM for the binding of SRP to the SecYEG-FtsY complex.
Figure 6.

SecYEG-FtsY complex formation in the presence of FtsQ-RNC, HemK-RNC, or SRP. (A) SecYEG-FtsY complex formation in the presence of FtsQ-RNC. MDCC-labeled SecYEG in nanodiscs was titrated with FtsY(Bpy) in the presence of increasing concentrations of FtsQ-RNC (µM): 0, ●; 0.005, ○; 0.01, ■; 0.02, □; 0.05, ▲; 0.1, △. For clarity, representative error bars (SEM; n = 2) are indicated only on the last titration point. Kd values of ∼0.18 µM were determined by nonlinear fitting using equation 1 (Materials and methods); at concentrations of FtsQ-RNC >0.02 µM, the fluorescence change was too small to allow for the estimation of reliable Kd values. (B) Effect of FtsQ-RNC binding on the SecYEG-FtsY complex. Kd values from A are plotted against the concentration of FtsQ-RNC (○; right Y-axis). Donor (MDCC) fluorescence measured at saturation with FtsY(Bpy) (●, left Y-axis) is plotted relative to the initial fluorescence of SecYEG(MDCC) measured before the addition of FtsY(Bpy); error bars represent SEM from (A). Nonlinear fitting to equation 2 (Materials and methods) yielded an apparent Kd = 8 ± 1 nM for the binding of FtsQ-RNC to the SecYEG-FtsY complex. (C) SecYEG-FtsY complex formation in the presence of HemK-RNC. Titrations were performed as in A in the presence of increasing concentrations of HemK-RNC (µM): 0, ●; 0.03, ○; 0.08, ■; 0.25, □. (D) No effect of HemK-RNC on SecYEG-FtsY complex formation. (E) SecYEG-FtsY complex formation in the presence of SRP. MDCC-labeled SecYEG was titrated with FtsY(Bpy) in the presence of increasing concentrations of SRP (µM): 0, ●; 0.05, ○; 0.1, ■; 0.2, □; 0.3, ▲; 0.5, △. Apparent Kd values of ∼0.2 µM (○, right Y-axis) were determined by nonlinear fitting as in A. (F) Effect of SRP on the SecYEG-FtsY complex. Kd values from C are plotted against the SRP concentration (○). Donor (MDCC) fluorescence measured at saturation (●, left Y-axis) is plotted relative to the initial fluorescence measured before the addition of FtsY(Bpy). Nonlinear fitting as in B yielded an apparent Kd of 40 ± 10 nM for the binding of SRP to the SecYEG-FtsY complex.

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