Fig. 2.

Functional properties of GroEL^WT and the GroEL^D83A/R197A. (A) Steady state hydrolysis of ATP as a function of [ATP] by GroEL^WT (blue) or GroEL^D83A/R197A (red) measured as previously described in the absence (open dots) or presence (filled dots) of denatured α-lactalbumin (3.6 μM). These measurements were conducted at 37 °C with 100 mM K⁺ and 10 mM Mg²⁺. (B) Presteady state hydrolysis of ATP by GroEL^WT (blue) or GroEL^D83A/R197A (red) monitored by fluorescence of phosphate binding protein labeled with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide. The accepting state complex [^cisGroEL-ADP-GroES][^transGroEL] was mixed with ATP and labeled PBP to initiate the reaction. The rate constant for the burst phase was 0.49 s⁻¹ (GroEL^WT) and 0.45 s⁻¹ (GroEL^D83A/R197A). These measurements were conducted at 37 °C with 200 mM K⁺ and 10 mM Mg²⁺. (C) Dissociation of GroES from the cis ring of GroEL^{E315C-IAEDANS} (blue) or GroEL^{D83A/R197A/E315C-IAEDANS} (red) monitored by decrease of FRET signal. The resting state complex [^cisGroEL^IAEDANS-ADP-GroES^F5M][^transGroEL^IAEDANS-ADP] was mixed with ATP and the excess amount of unlabeled GroES to initiate the reaction. The mean residence of GroES^F5M dissociating from the wild type GroEL^IAEDANS resting state was ∼12 s and the D83A/R197A GroEL^IAEDANS resting state was ∼37 s. These measurements were conducted at 37 °C, 200 mM K⁺, and 10 mM Mg²⁺.