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

Walker, Thomas E.; Shirzadeh, Mehdi; Sun, He Mirabel; McCabe, Jacob W.; Roth, Andrew; Moghadamchargari, Zahra; Clemmer, David E.; Laganowsky, Arthur; Rye, Hays S.; Russell, David H.

doi:10.26434/chemrxiv-2021-vk20s

Cited by 6 publications

(10 citation statements)

References 44 publications

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“…The combination of statistical thermodynamic modeling and native MS to extract and quantify allosteric parameters from the partition function is powerful but has limitations. The most obvious is that while mechanistic models can be readily envisioned for lattices in which nearest-neighbor interactions can be expected to be dominant, such as ring-shaped proteins, it is less straightforward to develop such models for hetero-oligomeric proteins, or homo-oligomers with non-cyclic symmetry elements (e.g., double-rings like GroEL 6,44 and the proteosome 45 ). Another limitation has to do with the degeneracy of liganded states: e.g., for the NN-add model, the measured populations with half-filled 12mers convolve 924 individual configurations distributed among six distinct statistical weights ( Equation 2 ).…”

Section: Discussionmentioning

confidence: 99%

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

Norris

Lichtenthal

et al. 2022

Preprint

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Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric tryptophan-binding protein TRAP. First, we develop nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites ΔGN0, and for coupling between one or both adjacent sites, ΔGN1 and ΔGN2. Using the resulting partition function (PF) we explore the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored ligand-dependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (nMS). By resolving species with differing numbers of bound ligands by their mass, nMS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. The combination of statistical thermodynamic modeling with native mass spectrometry may provide the thermodynamic foundation for meaningful structure-thermodynamic understanding of cooperativity.

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Section: Discussionmentioning

confidence: 99%

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

Norris

Lichtenthal

et al. 2022

Preprint

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“…Native mass spectrometry (MS) has the potential to distinguish allosteric mechanisms by more directly probing bound-state distributions. 7,8,[20][21][22] Unlike measurements of average fractional saturation, native MS can directly probe the populations of states with different numbers of bound ligands. 22 The mass (and mass-to-charge ratios, m/z) of a macromolecular complex is exactly determined by the molecular weight of its constituents.…”

Section: Native Mass Spectrometry Can Measure Population Distributionsmentioning

confidence: 99%

“…Native mass spectrometry (MS) has the potential to distinguish allosteric mechanisms by more directly probing bound‐state distributions 7,8,20–22 . Unlike measurements of average fractional saturation, native MS can directly probe the populations of states with different numbers of bound ligands 22 .…”

Section: Introductionmentioning

confidence: 99%

“…This difference in affinity arises through “allosteric” interactions between the ligand‐binding sites, which alter the free energy change upon additional ligand binding 4,5 . Such thermodynamic changes are most often associated with structural changes that accompany ligand binding, though changes in protein dynamics have also been implicated 6–8 . A large proportion of biological processes are regulated by oligomeric ligand‐binding proteins, from hemoglobin 9 to p97 ATPase 10,11 .…”

Section: Introductionmentioning

confidence: 99%

“…4,5 Such thermodynamic changes are most often associated with structural changes that accompany ligand binding, though changes in protein dynamics have also been implicated. [6][7][8] A large proportion of biological processes are regulated by oligomeric ligand-binding proteins, from hemoglobin 9 to p97 ATPase. 10,11 Therefore, to understand biological regulation we must first understand the linkage between ligand-induced structural changes and changes to protein-ligand free energy landscapes.…”

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confidence: 99%

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Thermodynamic coupling between neighboring binding sites in homo‐oligomeric ligand sensing proteins from mass resolved ligand‐dependent population distributions

Norris

Lichtenthal

et al. 2022

Protein Science

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Homo‐oligomeric ligand‐activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand‐binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring‐shaped homo‐oligomeric trp RNA‐binding attenuation protein (TRAP). First, we developed a nearest‐neighbor statistical thermodynamic binding model comprising microscopic free energies for ligand binding to isolated sites ΔG0, and for coupling between adjacent sites, ΔGα. Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored ligand‐dependent population shifts using conventional spectroscopic and calorimetric methods and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand‐dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure–thermodynamic linkage that drives cooperativity.

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Dissecting the structural heterogeneity of proteins by native mass spectrometry

2023

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A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

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

Cited by 6 publications

References 44 publications

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

Thermodynamic coupling between neighboring binding sites in homo‐oligomeric ligand sensing proteins from mass resolved ligand‐dependent population distributions

Dissecting the structural heterogeneity of proteins by native mass spectrometry

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