Fig. 1.
ATP binds rapidly to unliganded GroEL and to the trans ring of the GroEL/GroES/ADP complex. (A) Ribbons diagram of a portion of the equatorial domain of 1 GroEL subunit in the GroEL/GroES/ADP/AlFx complex (ref. 24; PDB ID code: 1PCQ) showing the position of F44. (B) Emission spectra of EL44-OG alone (black trace), mixed with 500 μM ADP (green), and mixed with 500 μM ATP (red). Excitation was at 496 nm. (C) Change in fluorescence of EL44-OG on stopped-flow mixing with ATP. The trace could be fit (white line) as the sum of 2 exponentials with the rate constants shown. The schematic indicates OG (green) in the equatorial domains. (D) Dependence of the faster rate of fluorescence change on ATP concentration. Rate constants from experiments as in C (black) and E (red) at different ATP concentrations are plotted as a function of ATP concentration, giving straight lines with slopes equal to the respective second-order rate constants for ATP binding, as shown. (E) Change in fluorescence of the EL44-OG/GroES/ADP complex on stopped-flow mixing with ATP. D represents ADP in the cis ring, GroES is colored gray, and ATP (red) is shown adjacent to the ring to which it binds. (F) As in E, except with unliganded EL44-OG. (G) As in E, but with MDH bound to the trans ring, represented as a blue line in the schematic. (H) Stopped-flow mixing experiment similar to that in E, except GroEL in the complex was labeled with OG on position 315 on the outside of the apical domains. Here, the trace could be fit as a single exponential with the indicated rate. (I) Stopped-flow experiment using the single-ring version of GroEL, SR1, labeled with OG on a cysteine substituted at position 315 (see Table S1 for a summary of rates and amplitudes.)