Allostery Without Conformational Change: A Native Mass Spectrometry Perspective

Native electrospray ionization mass spectrometry (nESI-MS) enables studies of intact proteins, protein complexes, and protein-protein 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 – 100 ºC, 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 for GroEL single ring mutant (SR1). Temperature-dependent shifts for average charge states (Zavg) and rotationally-average collision cross sections (CCS) for both apo- and nucleotide-bound SR1 complexes (SR1-ADPn, n = 1-7) indicate that nESI buffers alter structure, stabilities and dynamics. These studies report nucleotide (ADP) binding affinities (Ka) 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 ºC), but cooperativity is significantly diminished at higher temperatures (21 – 31 °C). While cooperative ADP binding is only observed at low temperatures (4 ºC) for TEAA solutions, it is absent in EDDA solutions. Collectively, these findings illustrate very different influences of ammonium and alkyl ammonium ions on the conformation and dynamics of SR1 as manifested by changes in Zavg and thermodynamics for nucleotide binding. Moreover, temperature dependent changes in Zavg and ligand binding provide additional experimental data that supports prior work on the effects of hydration on cold protein folding and recent computational results for the effects of hydration water on protein binding sites as well as membrane protein complex-lipid binding.