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

Abstract

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.

Surface Activity of Amines Provides Evidence for the Combined ESI Mechanism of Charge Reduction for Protein Complexes

“…A recent example reported by Walker et al revealed remarkable effects of native MS buffers, AmAc and EDDA, on the conformation and stabilities of the GroEL tetradecamer. 42 The protein complexes of C-reactive protein (CRP of ∼115 kDa), pyruvate kinase (PK of ∼237 kDa), and glutamate dehydrogenase (GDH of ∼334 kDa (experimental)) were studied using mixed buffers containing various concentrations of AmAc and TEAA, and the effects of nESI (nano-ESI) emitter potentials were also examined. Higher concentrations of TEAA and higher emitter potentials at each TEAA concentration decreased the Z avg for all proteins.…”

“…Monomeric proteins have been extensively studied via ESI-MS; therefore, we have chosen to conduct these experiments on protein complexes, which offer more diverse and less well-studied responses to charge reduction conditions. A recent example reported by Walker et al revealed remarkable effects of native MS buffers, AmAc and EDDA, on the conformation and stabilities of the GroEL tetradecamer . The protein complexes of C-reactive protein (CRP of ∼115 kDa), pyruvate kinase (PK of ∼237 kDa), and glutamate dehydrogenase (GDH of ∼334 kDa (experimental)) were studied using mixed buffers containing various concentrations of AmAc and TEAA, and the effects of nESI (nano-ESI) emitter potentials were also examined.…”

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

“…Mg 2+ ions are essential for the binding of ATP to proteins and K + ions have been known as strong activators of ATPase activity. In previous work we have shown that the Mg 2+ concentration impacts the stoichiometry of GroES binding to GroEL 21 (also see Figure S4 ), which illustrates the importance of Mg 2+ concentration for proper functioning of the GroEL nanomachine. Lorimer et al have shown that K + ions are necessary for the modulation of the ATPase mechanism of GroEL.…”

“…1 While the structure, dynamics, and ATP binding of GroEL has been extensively investigated, [8][9][10][11][12][13][14][15][16][17][18][19][20] we recently reported results using native mass spectrometry (nMS) that reveal new insights about stability and stoichiometry of GroEL-ATP/GroES interactions. 21 Using variable-temperature electrospray ionization (vT-ESI) we found that GroEL-ATP binding was temperature and ATP-concentration dependent, indicating that the thermodynamics of this vital interaction for the GroEL nanomachine might reveal new information as to how the GroEL nanomachine operates. This could provide new strategies for analysis of thermodynamics of individual binding reactions for multi-dentate systems which is especially challenging and remains difficult to achieve with current methods.…”

Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Toward the Conformational Dynamics of Mega Dalton Protein Aggregates

“…It has been used to analyze a vast number of proteins and oligonucleotides and has provided great insight into their structural and functional characteristics, which could not be readily obtained by other techniques . For example, native MS has been used to investigate peptide interactions, DNA thermodynamics, , antibody binding, chaperones, , protein aggregation, , as well as very large biomolecular complexes such as ribosomes and intact viruses . This is due to the development of electrospray ionization (ESI), which has allowed researchers to transfer biomolecules from solution into the gas phase without inducing significant disruptions to their structure.…”