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

Abstract

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.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions

“…The approach has also uncovered that specific protein-lipid interactions can allosterically modulate other interactions with protein, lipid, and drug molecules ( Marcoux et al, 2013 ; Yen et al, 2018 ; Cong et al, 2017 ; Patrick et al, 2018 ; Bolla et al, 2018 ; Gault et al, 2016 ). More recently, native MS has proved useful in dissecting the thermodynamics of individual nucleotide binding events to GroEL, a 801 kDa tetradecameric chaperonin ( Walker et al, 2023 ). In contrast to traditional approaches, such as ITC and SPR, native MS can resolve and dissect individual binding events enabling the measurement of binding thermodynamics, which is of paramount importance to understanding the molecular driving forces of non-covalent interactions.…”

“…The technique is capable of maintaining non-covalent interactions and native-like structure in the gas phase ( Ruotolo et al, 2005 ; Laganowsky et al, 2014 ), essential for studying biochemical interactions with small molecules, such as the binding of drugs, lipids, and nucleotides ( Laganowsky et al, 2014 ; Allison et al, 2015 ; Barrera et al, 2008 ; Campuzano et al, 2019 ; Gupta et al, 2017 ; Marcoux et al, 2013 ; Yen et al, 2018 ; Zhou et al, 2011 ). In combination with a variable temperature nano electrospray ionization device, native MS has determined the thermodynamics for protein-protein and protein-ligand interactions ( Daneshfar et al, 2004 ; Deng et al, 2013 ; Raab et al, 2020 ; Walker et al, 2023 ; Qiao et al, 2021 ; McCabe et al, 2021 ). For example, the molecular interaction between the signaling lipid 4,5-bisphosphate phosphatidylinositol and Kir3.2 is dominated by a large, favorable change in entropy ( Qiao et al, 2021 ).…”

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions

“…33,34,48,49,62,63 More recently, native MS has proved useful in dissecting the thermodynamics of individual nucleotide binding events to GroEL, a 801 kDa tetradecameric chaperonin. 39 In contrast to traditional approaches, such as ITC and SPR, native MS can resolve and dissect individual binding events enabling the measurement of binding thermodynamics, which is of paramount importance to understanding the molecular driving forces of non-covalent interactions.…”

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II

“…Variable-temperature electrospray ionization (vT-ESI), in which the solution temperature is varied during electrospray, has been used to determine the T m values of proteins, [34][35][36][37][38][39] protein complexes, 19,34,[40][41][42][43][44][45][46] protein as well as DNA complex unfolding pathways, 42,[47][48][49] and the thermochemistry of ligand-binding to protein-and DNA-ligand complexes. 38,47,50,51 In combination with ion mobility spectrometry, thermochemical values can be determined for individual groups of closely related protein conformers. 36,38,40,52,53 The T m values of 7 analyte molecules from a mixture of proteins was determined simultaneously, 54 clearly demonstrating the multiplexing capacity of this technique.…”