Fluorescence correlation spectroscopy and allostery: The case of GroEL

Fluorescence correlation spectroscopy (FCS) is an experimental technique in which the equilibrium fluctuations of the fluorescent signal of molecules diffusing through a focused laser beam are measured. An autocorrelation analysis of these fluctuations provides information on dynamic processes, such as allosteric transitions, that the molecules undergo provided that they are fast relative to the diffusion time through the beam. In cases when the dynamics are slow relative to the diffusion time through the beam, FCS curves can yield information about the number of conformational states and their relative populations. Hence, FCS can be used to investigate allosteric systems with either slow or fast dynamics but the type of information gained in these two situations is different. Here, the utility of the FCS technique is exemplified in the case of the single-ring version of the Escherichia coli molecular chaperone GroEL that interconverts with relatively slow dynamics between two allosteric states: a T state with low affinity for ATP and an R state with high affinity for ATP. Thermodynamic analysis suggests that the T-state population should become negligible with increasing ATP concentrations, in conflict with the requirement for conformation cycling, which is essential for the operation of molecular machines. Surprisingly, FCS experiments showed that, 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.