Elucidation of the Molecular Mechanisms of Protein Folding

The chaperonin GroEL from Escherichia coli, a tetradecameric protein complex consisting of two heptameric rings stacked back to back with a central cavity, is one of the best characterized molecular chaperones that facilitate protein folding in vivo. The ATP– dependent control of the affinity for its target protein and the co-chaperonin GroES is essential for its molecular chaperone function, and this control occurs through a series of cooperative allosteric transitions of GroEL induced by MgATP2–. The equilibria and kinetics of the allosteric transitions of GroEL have thus been studied for some time by a variety of techniques. However, the initial bimolecular step of MgATP2– binding to GroEL, which must precede the allosteric transitions, remains to be clarified. Here, we studied the equilibrium and kinetics of MgATP2– binding to a variant of GroEL, in which Tyr485 was replaced by tryptophan, via isothermal titration calorimetry (ITC) and stopped-flow fluorescence spectroscopy (Figures 1 and 2). In the absence of K+ at 4 ~ 5 °C, the allosteric transitions and the subsequent ATP hydrolysis by GroEL are halted, and hence, the stopped–flow fluorescence kinetics induced by rapid mixing of MgATP2– and the GroEL variant solely reflected MgATP2– binding, which was well represented by bimolecular noncooperative binding with a binding rate constant, kon, of 9.14 × 104 M–1 s–1 and a dissociation rate constant, koff, of 14.2 s–1, yielding a binding constant, Kb (= kon/koff), of 6.4 × 103 M–1. We also successfully performed ITC to measure Figure 1. The structure around the MgATP2–-binding site of GroEL (a), the SAXS patterns of wild-type GroEL in the different allosteric states (b), and the binding kinetics of MgATP2– to GroEL(Y485W) (c and d).