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

Abstract

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.

Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1

“…These limitations may in some cases be overcome by combining charge-reducing reagents to provide greater separation between charge states. Charge-reducing reagents can also alter and/or compete with the formation of desired complex or reduce the stabilities of the complexes. − Such peak broadening compromises our capabilities to mass-select individual charge states for structural analysis using tandem mass spectrometry (MS) ( vis . collision-induced dissociation (CID), surface-induced dissociation (SID), electron capture dissociation (ECD), , ultraviolet photodissociation (UVPD), and/or ion mobility-MS).…”