Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding

Abstract

Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A-ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.

Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains

“…By following the gas phase unfolding pattern of protein ions, the unfolding “heat maps” [41] are a function of protein higher order structure and protein-ligand binding state [39, 42, 53–55]. Both intact OCP and NTD undergo gas phase unfolding to increase their CCS, whereas CTD remarkably undergoes no significant unfolding (see Figure 5 for maps of intact OCP monomer, dimer, OCP NTD and CTD).…”

“…We analyzed OCP functional domains (NTD and CTD) from partial digestion of active OCP by using IM and collisional unfolding [39] under native MS conditions. Two OCP functional domains were compared with inactive intact OCP.…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…When calibrated with proteins of known structures, collected data allow the calculation of the average collisional cross section of a protein (in Å 2 ) providing important 3D structural information. In addition, these data enable the calculation of dissociation constants of binding partners as well as the revelation of subtle conformational changes and ligand binding-induced unfolding patterns (Laganowsky et al, 2014;Niu & Ruotolo, 2015;Rabuck et al, 2013;Stojko et al, 2015). Thus, by IM-MS information is gathered on the 3D structure and on the dynamics of a protein.…”

Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

“…Drift times (DTs) provide information on conformational dynamics [13, 14], and folding/unfolding intermediates [15, 16]. Native MS and IM report on ligand-induced stability and conformational changes for protein-ligand and protein-protein interactions [17, 18]. …”

“…Such CIU experiments were first described for small monomeric protein ions [19], but they are now applied for stabilities and conformational changes even with ligand binding [20]. CIU can also report on the consequences of small-molecule attachments to large protein systems [21, 22], revealing cooperative binding mechanisms, overall stability of multi-protein systems [23], and antibody-isoform differentiation of disulfide bonding and glycosylation levels [24]. Software advances for CIU are allowing comparison between ions with subtle differences [25].…”