Abstract
Native electrospray ionization-mass spectrometry (nESI-MS) enables studies of intact proteins, protein complexes, and protein-ligand complexes. Variable temperature (vT)-nESI-MS, where the temperature of the solution contained in the ESI emitter can be varied from 2 to 100 °C, adds new capabilities for dissecting the thermodynamics for protein-ligand binding. Here, vT-nESI-MS and ion mobility spectrometry (IMS) are used to compare the effects of temperature and nESI buffers on nucleotide (ADP) binding for the GroEL single ring mutant (SR1). Temperature-dependent shifts for average charge states (Z(avg)) and rotationally averaged collision cross sections (CCS) for both apo- and nucleotide-bound SR1 complexes (SR1-ADP(n), n = 1-7) indicate that nESI buffers alter structure, stabilities, and dynamics. These studies report nucleotide (ADP) binding affinities (K(a)) and insight into cooperativity and enthalpy-entropy compensation (EEC). Specifically, we focus on three commonly used native ESI buffers: ammonium acetate (AmAc), triethylammonium acetate (TEAA), and ethylenediammonium acetate (EDDA). In AmAc solutions, ADP binding is highly cooperative at low temperatures (2-21 °C) but is significantly diminished at higher temperatures (21-31 °C). While cooperative ADP binding is only observed at low temperatures (4 °C) for TEAA solutions, it is absent in EDDA solutions. Collectively, these findings illustrate very different influences of ammonium and alkyl ammonium ions on the SR1 conformation and dynamics as manifested by changes in Z(avg) (change of solvent-accessible surface area) and thermodynamics for nucleotide binding. Moreover, temperature-dependent changes in Z(avg) and ligand binding provide additional experimental data that support prior work on the effects of hydration on cold protein folding. These results also align with recent computational work for the effects of hydration water on protein binding sites as well as membrane protein complex-lipid binding. The observed temperature-dependent changes in Z(avg), buffer-dependent nucleotide binding, EEC, and changes in heat capacity strongly suggest that ADP influences the conformational states of the SR1 complex. Note, however, that large-scale structural changes in the SR1 complex are not observed in the IMS CCS experiments. Collectively, these results suggest that ADP binding alters key structural and/or dynamic properties of SR1, changes that are not observed in the overall, macroscopic structure of the complex. We suggest that SR1-ADP binding is an archetypal example of "allostery without (measurable) conformational change".