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 GroEL 14 and GroES 7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (T m ∼ 70 °C). This destabilization is accompanied by temperaturedependent reaction products that have previously unreported stoichiometries, viz. GroEL 14 −GroES y −ATP n , where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg 2+ 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 Mg 2+ and ATP concentrations on the stoichiometry of the GroEL−GroES complex, with Mg 2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL−GroES complex (GroEL 14 −GroES 7 vs GroEL 14 −GroES 8 ) 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.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, 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 evidences for de-stabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the pre-viously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry re-veals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiome-try of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temper-ature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the utility of native MS for investiga-tions of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

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−ATP 7 and GroEL−ATP 14 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.; et al.

Biomolecules exist in the ever-changing environment of solution, which defines their structure, stability, dynamics, and function. Moreover, these effects are manifested in protein folding, protein-protein, and protein-ligand interactions. Thermodynamic studies, especially changes in heat capacity (DCp), are used to monitor the effects of solution temperature, concentration, ligands and/or co-solutes, but it can be challenging to relate such changes to molecular level reactions. Native mass spectrometry (nMS) has emerged as a complimentary technique to the traditional methods of structural and mechanistic characterization of biomolecules. Coupling of variable-temperature electrospray ionization (vT-ESI) to nMS affords mapping these changes to specific reactants and/or products using m/z-dispersion, whereas traditional thermodynamic measurements provide an ensemble-average of products. Here, we utilize vT-ESI-nMS to quantitate the thermodynamic contributions of stepwise binding of individual ATP or ADP ligands to GroEL-a tetradecamer chaperonin complex capable of binding up to 14 ATP molecules. We also show that small ions (viz. NH4 + ) are important contributors to the binding mechanisms of ATP and ADP to GroEL tetradecamer. The thermodynamic measurements reveal extensive enthalpy-entropy compensation (EEC) as well as increased cooperative effects for the formation of GroEL-ATP14, whereas similar cooperativity for ADP binding is absent. The thermodynamic data demonstrate that ATP binding in the cis ring (GroEL-ATP1-7) is largely entropic compared with more enthalpically driven reactions for ATP binding to the trans ring (GroEL-ATP8-14), owing to negative inter-ring cooperativity. The overall entropic effects for ATP binding to GroEL tetradecamer are attributed to conformational changes of the GroEL tetradecamer, but the magnitude of the entropy is also attributable to reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity. This study reveals new pathways, viz. nMS, for experimental studies aimed at expanding our understanding of biologically relevant chaperonin functions.