Cooperative Salt Bridge Stabilization of Gas-Phase Zwitterions in Neutral Arginine Clusters

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that dimeric Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS (MS/MS-IMMS) coupled with increasing collisional activation potentials. For six of the seven SOD1 variants, increasing activation promoted dimers to transition through two unfolding events to access three gas-phase conformers before dissociating symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. We localise this effect to the formation of a new salt-bridge in the first activated conformation. To examine the data quantitatively, we generated a model of SOD1 gas phase unfolding and dissociation, and applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals an increase in the barrier height to unfolding in G37R to be >5 kJ/mol-1 higher than for the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates the importance of bond formation during the unfolding of proteins in vacuum, and provides a framework for comparing quantitatively the free energy landscape they explore upon activation.

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The Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase Through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt-Bridge Formation

10.26434/chemrxiv.7898246.v2 ◽

2019 ◽

Author(s):

Luke McAlary ◽

Julian Harrison ◽

J. Andrew Aquilina ◽

Steven Fitzgerald ◽

Celine Kelso ◽

...

Keyword(s):

Superoxide Dismutase ◽

Free Energy ◽

Gas Phase ◽

Protein Unfolding ◽

Energy Landscape ◽

Salt Bridge ◽

Native Mass Spectrometry ◽

Free Energy Landscape ◽

Related Point ◽

Macromolecular Protein

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that dimeric Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS (MS/MS-IMMS) coupled with increasing collisional activation potentials. For six of the seven SOD1 variants, increasing activation promoted dimers to transition through two unfolding events to access three gas-phase conformers before dissociating symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. We localise this effect to the formation of a new salt-bridge in the first activated conformation. To examine the data quantitatively, we generated a model of SOD1 gas phase unfolding and dissociation, and applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals an increase in the barrier height to unfolding in G37R to be >5 kJ/mol-1 higher than for the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates the importance of bond formation during the unfolding of proteins in vacuum, and provides a framework for comparing quantitatively the free energy landscape they explore upon activation.

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Salt Bridge Chemistry Applied to Gas-Phase Peptide Sequencing: Selective Fragmentation of Sodiated Gas-Phase Peptide Ions Adjacent to Aspartic Acid Residues

Journal of the American Chemical Society ◽

10.1021/ja973467r ◽

1998 ◽

Vol 120 (13) ◽

pp. 3188-3195 ◽

Cited By ~ 112

Author(s):

Sang-Won Lee ◽

Hyun Sik Kim ◽

J. L. Beauchamp

Keyword(s):

Gas Phase ◽

Aspartic Acid ◽

Salt Bridge ◽

Peptide Sequencing ◽

Peptide Ions

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Gas-phase structures of solution-phase zwitterions: Charge solvation or salt bridge?

International Journal of Mass Spectrometry ◽

10.1016/j.ijms.2008.12.011 ◽

2009 ◽

Vol 281 (1-2) ◽

pp. 97-100 ◽

Cited By ~ 14

Author(s):

Miriam K. Drayß ◽

Dirk Blunk ◽

Jos Oomens ◽

Nick Polfer ◽

Carsten Schmuck ◽

...

Keyword(s):

Gas Phase ◽

Salt Bridge ◽

Solution Phase ◽

Phase Structures ◽

Charge Solvation

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Ion-Ion interactions affect (re)folding dynamics of ubiquitin ions in the gas phase. Distinct folding intermediates formed via charge-reduction pathways

10.26434/chemrxiv.5678872.v1 ◽

2017 ◽

Author(s):

Jacquelyn R. Jhingree ◽

Perdita E. Barran

Keyword(s):

Electron Transfer ◽

Gas Phase ◽

Cross Sections ◽

Structural Integrity ◽

Salt Bridge ◽

Charge Reduction ◽

Net Charge ◽

Ion Interactions ◽

Folding Dynamics ◽

Low Charge

Using Ubiquitin as an exemplar protein we examine the effect of net charge reduction post electrospray ionisation by exposure to the electron transfer reagent, 1,3-dicyanobenzene. We monitor the change in gas phase conformation of both precursor and products with ion mobility mass spectrometry (IM-MS). Dramatic conformational rearrangement is seen for low charge state ions upon exposure to the electron transfer reagent. Ions with low charge states sprayed from both native-like and denaturing solvent conditions undergo structural transitions to conformers with cross sections in the range measured for the native structure (TWCCSN2®He, 950-1000 Å2). Thus, we infer that some memory of the solution phase structure is retained in the gas phase. Intermediate structures are seen in the reduction of the [M+6H]6+ ion sprayed from both native-like and denaturing solvents. Further, the reduction pathway of this ion shows compaction to structures with a TWCCSN2®He centred at 1069 Å2 (5+) and 949 Å2 (4+) for ions originating from native-like and denaturing solvents respectively. We propose that charge reduction sites for radical anion localisation (to effect electron transfer) are not easily accessible in the case of ubiquitin molecules originating from native-like solution conditions. This highlights the importance of salt bridge interactions in maintaining the structural integrity of a protein in the gas phase. Most interestingly, two distinct conformer populations are seen for the 6+ charge-reduced product originating from the 7+ and, the 6+ exposed to radical anions (post ESI); we infer that these populations are intermediate in the refolding of ubiquitin in the gas phase, sometimes transient. Overall, we are able to monitor the refolding pathway of ubiquitin in the gas phase as its charge is reduced and show that charge-charge interactions play a significant role in the gas phase conformation adopted; whereby specific conformations are formed.

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