Fig. 2.

Relatively slow binding of a nonnative substrate, DM-MBP, to unliganded GroEL and to the trans ring of the GroEL/GroES/ADP complex. (A) Emission spectrum of DM-MBP labeled with CPM (donor) while bound to GroEL (D, black trace), emission spectrum of EL44-OG (acceptor) while complexed with nonnative DM-MBP (A, blue trace), and emission spectrum of the complex of DM-MBP-CPM with EL44-OG (D-A, red trace), all excited at 384 nm, the excitation maximum for CPM. (B) Change in donor channel fluorescence on stopped-flow mixing of nonnative DM-MBP-CPM with EL44-OG. Blue line represents nonnative DM-MBP, and D in the yellow circle represents the CPM label. (C) Dependence of the faster rate constant for DM-MBP binding on GroEL concentration. Rate constants from experiments as in B at different concentrations of GroEL (black) or from similar experiments using the GroEL/GroES/ADP complex (red) are plotted vs. GroEL concentration, yielding straight lines with slopes (second-order rate constants) of 6.11 × 10⁶ M⁻¹s⁻¹ and 5.36 × 10⁶ M⁻¹s⁻¹, respectively. (D) Change in donor fluorescence on stopped-flow mixing of nonnative DM-MBP-CPM with the EL44-OG/GroES/ADP complex and 500 μM ATP. In multiple experiments (n = 6), the rate of the faster phase was consistently slightly faster for the trans ring in the presence of ATP than in its absence: 2.08 ± 0.11 vs. 1.36 ± 0.22 (Table S2).