Bound Anions Differentially Stabilize Multiprotein Complexes in the Absence of Bulk Solvent

Abstract

The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility-mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting ‘shell’ of counter-ions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility-mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of twelve different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counter-ions on gas-phase protein structure and allows us to rank and classify counter-ions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride and nitrate anions are amongst the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion-protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counter-ion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high-affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent.

Bound Cations Significantly Stabilize the Structure of Multiprotein Complexes in the Gas Phase

“…In addition, the relative stability of the five complexes studied here are not influenced by cation additives, with BLA requiring the most energy to dissociate and TTR requiring the most energy to unfold under equivalent conditions. [9] Despite these similarities, we find several significant differences in the stabilization provided by cation additives when compared with our previous anion data. Firstly, cation adducts seem to stabilize protein complexes against CID to a greater extent, on average, than equivalent anions.…”

“…This further indicates a disparity between the stabilization mechanisms operative for anionic and cationic additives. [9] The above differences between anionic and cationic stabilizers inform our mechanistic description of their action, which has been normalized for the relative binding affinities of the cations studied here. Whereas anions perform optimally as stabilizers when they bind to the protein and then dissociate from the complex after relatively minimal activation, the best cationic stabilizers are those that remain bound to the protein assembly in large numbers, even following extensive activation in the gas phase.…”

“…We observe that, in general, cations of high charge-per-unit-area stabilize proteins in a complimentary and significantly different way relative to most anions, [9] and we plan to exploit this in the future by using salt additives that are tailored to take advantage of both cationic and anionic protein adducts to improve protein structural stability. We believe that such additives are critical for IM-MS to fully-realize its potential as a high-throughput method for discovering multiprotein topology and structure, and as a means of elucidating the critical role of surfactant molecules in stabilizing gas-phase membrane protein complexes.…”

“…[10] Our previous data revealed that anions bind to protein complexes during or prior to the nano-electrospray ionization (nESI) process and can stabilize protein ions through dissociation as neutrals, which act to carry away excess energy from the gas-phase protein ions, thus allowing their structures to remain compact -in configurations easily correlated to X-ray and NMR datasets. [9] In this work, we study the influence of cation-based stabilizers, compare these additives to our previous anion dataset, and find dramatic mechanistic differences between the two.Figs. 1A and 1B show data for tetrameric transthyretin (TTR, 55 kDa).…”

“…We have focused on the former, using Hofmeister-type salt additives, and have recently classified a large number of anions for their ability to stabilize multiprotein structure [9] using measurements of both collision induced unfolding (CIU), where ions are heated with collisions and induced to unfold, and collision induced dissociation (CID), where increased collisional heating leads to the dissociation of assemblies into a highly unfolded monomers and stripped complexes. [10] Our previous data revealed that anions bind to protein complexes during or prior to the nano-electrospray ionization (nESI) process and can stabilize protein ions through dissociation as neutrals, which act to carry away excess energy from the gas-phase protein ions, thus allowing their structures to remain compact -in configurations easily correlated to X-ray and NMR datasets.…”