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

Abstract

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. File list (2) download file view on ChemRxiv Main Text.pdf (1.37 MiB) download file view on ChemRxiv Supplementary Information.pdf (1.37 MiB)

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology

“…Hybrid MD–Monte Carlo approaches have been developed for the combined search of conformer and charge-isomer space in the gas phase. These indicate that side chains have a propensity to fold onto the protein surface with consequent structure contraction and formation of new hydrogen bonds and salt bridges, a prediction for which experimental evidence is emerging . Such structural rearrangements promote self-solvation and are compatible with maintenance of a native-like fold.…”

“…These indicate that side chains have a propensity to fold onto the protein surface with consequent structure contraction and formation of new hydrogen bonds and salt bridges, 70 a prediction for which experimental evidence is emerging. 73 Such structural rearrangements promote selfsolvation and are compatible with maintenance of a native-like fold. An interesting feature in the emerging picture of folded protein ions in the gas phase is their ability to compensate for the energetic penalty of charge separation in vacuo with favorable, conformation-specific intramolecular interactions, in line with growing experimental and theoretical evidence.…”

Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions

“…For example, a superoxide dismutase mutant containing an additional arginine exhibits altered stability in IM-MS due to the formation of an additional salt bridge after desolvation. 23 In solution, alkaline pH unfolds proteins by breaking salt bridges and H-bonds and causing coulombic repulsion due to the high density of negative charges. In positive ionization mode, the negative charges are neutralized mitigating their repulsive effect.…”

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

“… The fact that proteins lacking basic residues favor the CRM over CEM reveals the limitations of using charge state signatures to assess the distribution of folded states . As a result, proteins with few ionizable groups may display diverging charging patterns upon unfolding. Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. ,,, Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures. Gas-phase unfolding is routinely used to identify compounds that can stabilize protein complexes by occupying specific binding pockets. ,− We find that moving charged sites can have a dramatic effect on a protein’s conformation in the gas phase.…”

“… Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. 9 , 15 , 54 , 55 Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures.…”