Oligomeric, ring-shaped nano-machines that are fueled by ATP are ubiquitous in all three kingdoms of life and are involved in a wide range of processes that include, for example, protein folding, protein degradation, DNA and RNA remodeling, and protein insertion into membranes (for review, see ref. 1). These assemblies are true machines because they carry out work by undergoing movements that are coordinated in time and space and are driven by energy consumption (i.e., ATP binding and hydrolysis). One fundamental question regarding such machines relates to the coupling between the order of their ATPpromoted conformational changes ( Fig. 1) and their biological function. These conformational changes can take place in a concerted fashion according to the MonodWyman-Changeux (MWC) model (2), as proposed and recently demonstrated in the case of the chaperonin GroEL (3, 4). Alternatively, the changes can occur in a domino-like sequential fashion according to the Koshland-Némethy-Filmer model (5) or in a probabilistic manner, as suggested in the case of the proteolytic machine ClpP (6). A second fundamental question regarding such machines concerns the mechanisms by which ATP consumption is converted into work. Both questions are elegantly addressed, in the case of the chaperonin GroEL, in the report by Corsepius and Lorimer (7) published in PNAS.GroEL consists of two back-to-back stacked heptameric rings, with a cavity at each end in which protein folding can take place under confining conditions. GroEL assists protein folding by cycling between protein substrate acceptor and release states upon ATP binding and hydrolysis. ATP binding to GroEL occurs with positive cooperativity within rings and negative cooperativity between rings, both with respect to ATP. A nested allosteric model that accounts for these observations was put forward (3) in which each ring of GroEL switches in a concerted MWC fashion between a T state, with low affinity for ATP and high affinity for protein substrates, and an R state, with high affinity for ATP and low affinity for protein substrates. In the presence of ATP, the GroEL double-ring can, therefore, exist in three states: TT, TR, and RR. Because of the negative interring cooperativity, the TR→RR transition is less favored than the TT→TR transition, thus ensuring that the two rings can operate out of phase with each other.Complexes of protein substrates with GroEL are heterogeneous because bound nonfolded substrates can exist in multiple conformations that interact with different configurations of GroEL subunits (8). It has, therefore, been difficult to quantitate the effects of substrate binding on the allosteric properties of GroEL. Corsepius and Lorimer (7) have neatly circumvented this heterogeneity problem by labeling GroEL with tetramethylrhodamine (TMR) at position 242 in the apical domain near the protein substrate-binding site. This position was chosen so that TMR molecules attached to adjacent subunits can form noncovalently "stacked" dimers in the T state but not in the R stat...