Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

Abstract

The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol (m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared to the “native” complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a pre-channel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets where enrichment of the supercharging reagent during droplet evaporation occurs.

Do Charge State Signatures Guarantee Protein Conformations?

“…For large protein complexes (Figure 1c), experimental evidence supports a mechanism whereby manipulation of surface charge, by charge reduction [31] or enhancement [32], does not necessarily perturb the protein structure as evidenced for transthyretin [31], stable protein 1 [32], and a protective antigen prechannel complex [33]. This is contrary to the situation observed for concanavalin A in which the tetramer CCS, and solution phase tetramer to dimer equilibrium, was perturbed by the addition of m-NBA [33].…”

What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

“…Additive-associated charge increases for analytes sprayed from denaturing solutions have been attributed to increased surface tension (γ) [41], as predicted by extending the Rayleigh stability limit (equation 1) to the charged residue model [5]. $${\mathrm{normalZ}}_{\mathrm{normalR}}=8\pi {\left({\epsilon }_0\gamma {\mathrm{R}}^3\right)}^{1∕2}$$ In contrast, increases observed when native solutions are supplemented have been blamed on protein denaturation [47,48]. The mechanism of supercharging is debated, as are molecules classified as superchargers.…”

“…NBA and sulfolane addition to aqueous protein solutions had relatively little effect on structure at concentrations < 0.5% and < 1 M, respectively [53,58]. That structure could be lost by combining sulfolane addition with temperature elevation or guanidinum hydrochloride addition led to a proposal that sulfolane-associated charge increases reflect denaturation from superimposed thermal and reduced stability effects [58,60,48], making it relevant to consider what temperatures would be attainable by stable (non–evaporating) droplets moving through a bath of room temperature, atmospheric pressure laboratory air.…”

“…Unlike TWIMS, DMAs measure cross–sections directly, rather than inferring them from multipoint calibrations performed against known structures under identical conditions. Secondly, the DMA measures mobilities upstream of the MS interface, immediately following solvent evaporation, (thus still enduring the temperature elevations hypothesized by the Williams lab) [58,60,48], but prior to any collisional heating or declustering applied in the atmosphere–to–vacuum interface. Clearly, a trade–off for this latter benefit is that analyte ions may be solvated when mobility–analyzed, but subsequently desolvated by collisions in the atmospheric pressure–vacuum interface.…”

On the preservation of non-covalent protein complexes during electrospray ionization

“…The high concentration of SCA triggers the charge increase of protein ions as well as protein unfolding in charged microdroplets. Remarkably, the disruption of native protein complex conformations in supercharging experiments was observed even when nano-ESI emitters were used [41], for which the droplet lifetime is estimated to be in the range approximately 1-100 µs [21]. The occurrence of significant conformational changes in large protein complexes under certain experimental conditions in nano-ESI droplets may seem puzzling at first glance, because the unfolding of large proteins usually requires much longer than 1-100 µs.…”