The GroE chaperone system of E. coli is comprised of GroEL and GroES. GroEL consists of 14 identical ~57 kDa subunits arranged in two heptameric rings. It is a molecular machine that facilitates protein folding. During the folding cycle, the system undergoes dramatic conformational changes associated with the binding, hydrolysis and release of the nucleotides.

GroEL exists in at least two allosteric states, T and R, which interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns ‘on’, i.e. increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, ~50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the co-chaperonin GroES does the T-state population vanish.

Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be as slow as ~0.05 sec^-1. The slow release of hydrolysis products from the protein ensures that the cycling of this machine will continue even at high ATP concentrations. We suggest that slow product release may be a general property of molecular machines, setting them apart from regular enzymes. Namely, while enzymes are turnover-optimized, molecular machines are cycling-optimized.