A, Grp94 increases BiP FRET efficiency, indicative of a higher population of the BiP ADP conformation. The binding-defective mutant Grp94 K467A loses its capability to shift the BiP conformation. Solid lines are fits using Equation 1. B, kinetic FRET measurements show BiP transitioning from the ATP conformation to the ADP conformation (green circles). A similar experiment performed with Grp94 shows similar slow changes in FRET (red circles). C, binding kinetics between BiP and Grp94 is slow when BiP starts in the ATP conformation and transitions to the ADP conformation (red circles). In contrast, binding between BiP and Grp94 is fast when BiP starts in the ADP conformation (blue circles). Solid lines in B and C are single-exponential fits. D, kinetic model of conformation-specific binding between BiP and Grp94. Buffer conditions are the same as in Fig. 3. Error bars are the S.E. of the mean for at least three measurements. mP, millipolarization units.