SecYEG destabilizes the early intermediate in the RNC–SRP–FtsY complex. (A) Conformational states of the SRP–FtsY complex. Green and blue shapes denote FtsY and Ffh, respectively. T denotes GTP and D denotes GDP. (B) Equilibrium titration of the early complex in the absence (green) and presence of 0.05% DDM (black) or 24 µM SecYEG (red). The data were fit to Eq. 1 and gave the Kd values and FRET end points shown in F. (C) Effect of SecYEG on the stability of the early complex. The Kd values of the early complex were determined as in A at the indicated concentrations of SecYEG. (D) Equilibrium titration of the closed/activated complex was performed in the presence of 0.02% DDM (black) or 12 µM SecYEG (red), using 100 nM acrylodan-labeled SRP, 230 nM RNC, and 200 µM GppNHp. The data were fit to Eq. 1 and gave the Kd values shown in F. (E) Equilibrium titration of the activated complex was performed in the presence of 0.01% DDM (black) or 10 µM SecYEG (red), using 50 nM acrylodan-labeled FtsY C356, 300 nM RNC, and 200 nM GppNHp. The data points of the two titrations overlapped at 0.2 µM SRP, concealing the data point for the –SecYEG titration. The data were fit to Eq. 1 and gave the Kd values shown in F. (F) Summary of the Kd values and FRET end points from the experiments in B–E. The values are averages of two to four experiments ± SD. B–E show representative data from two to four experiments.
