The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod-Wyman-Changeux and sequential Koshland-Némethy-Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MSwhich provide the required resolution of ligand-bound states-and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.chaperonins | Hill coefficient M ultimeric proteins are often subject to allosteric regulation that is achieved by conformational changes induced or stabilized by ligand binding (1). Such allosteric regulation has been described by two classic models: (i) the Monod-WymanChangeux (MWC) model (2), in which conformational changes occur in a concerted manner and symmetry is conserved, and (ii) the Koshland-Némethy-Filmer (KNF) model (3), in which conformational changes take place in a sequential manner and symmetry is broken. In addition, it has been proposed more recently that conformational changes can take place in a probabilistic manner (4). The allosteric control of protein activity is frequently manifested in sigmoidal plots of initial reaction velocity or fractional saturation as a function of the ligand (substrate) concentration that indicates positive cooperativity in ligand binding. It has been impossible, however, to extract any mechanistic insights from these plots (5) because they only show how an average property of the ensemble (e.g., fractional saturation) changes with ligand concentration and do not reveal how the distribution of ligand-bound states changes with ligand concentration. Thus, for example, it is not possible to determine from such sigmoidal plots whether an allosteric transition takes place in a concerted MWC-like fashion (2) or via a sequential KNF-like mechanism (3). T...

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.

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The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis.Keywords: chaperonins; molecular chaperones; allostery; cooperativity; protein foldingThe GroE chaperonin system facilitates protein folding in an ATP-dependent manner (for reviews, see Horovitz et al. 2001;Thirumalai and Lorimer 2001;Saibil et al. 2002;Horovitz and Willison 2005). It consists of GroEL, an oligomer of 14 identical subunits of 57.3 kDa that form two stacked back-to-back heptameric rings (Braig et al. 1994) and its helper protein, GroES, which is a sevenmembered ring of identical subunits of 10 kDa (Hunt et al. 1996). Each subunit of GroEL is made up of three domains: (1) an apical domain that binds GroES and nonfolded protein substrates, (2) an equatorial domain that contains an ATP-binding site and is involved in inter-ring interactions, and (3) an intermediate domain that connects the apical and equatorial domains (Braig et al. 1994). GroEL belongs to a class of macromolecules collectively termed ''protein machines'' since it undergoes nucleoside triphosphate binding and hydrolysis-driven ordered conformational changes (Ranson et al. 2001) that are crucial for its function. These conformational changes are responsible for its cycling between protein substrate-binding and -release states (Staniforth et al. 1994;Yifrach and Horovitz 2000). ATP-triggered conformational changes in GroEL are reflected in steady-state kinetic measurements of initial rates of its ATPase activity at different concentrations of ATP, which have shown that it undergoes two ATP-induced allosteric transitions: one with a midpoint at relatively low ATP concentrations and the second with a midpoint at higher ATP concentrations (Yifrach and Horovitz 1995). Each of the allosteric transitions reflects homotropic intraring positive cooperativity in ATP binding. The higher ATP concentration required to induce the second allosteric transition reflects homotropic inter-ring negative cooperativity in ATP binding. A nested allosteric model that describes these results has been proposed (Yifrach and Horovitz 1995) in which each ring of GroEL interconverts in a concerted (Monod et al. 1965) manner between a T state, with high affinity for nonfolded proteins and low affinity for ATP, and an R state, with low affinity for nonfolded proteins and...