Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.

Dissecting the Thermodynamics of ATP Binding to GroEL One Nucleotide at a Time.

Variable-temperature electrospray ionization (vT-ESI) native mass spectrometry (nMS) is used to determine the thermodynamics for stepwise binding of up to 14 ATP molecules to the 801 kDa GroEL tetradecamer chaperonin complex. Detailed analysis reveals strong enthalpy-entropy compensation (EEC) for the ATP binding events leading to formation of GroEL-ATP7 and GroEL-ATP14 complexes. The observed variations in EEC and stepwise free energy changes of specific ATP binding are consistent with the well-established nested cooperativity model describing GroEL-ATP interactions, viz., intraring positive cooperativity and inter-ring negative cooperativity (Dyachenko A.; Proc. Natl. Acad. Sci. U.S.A.2013, 110, 7235-7239). Entropy-driven ATP binding is to be expected for ligand-induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play key roles. The capability for determining complete thermodynamic signatures (ΔG, ΔH, and -TΔS) for individual ligand binding reactions for the large, nearly megadalton GroEL complex expands our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate important new capabilities of vT-ESI-nMS for thermodynamic studies of protein interactions with ligands and other molecules such as proteins and drugs.