Niu Shuai, Ruotolo Brandon T
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109.
Protein Sci. 2015 Aug;24(8):1272-81. doi: 10.1002/pro.2699. Epub 2015 May 27.
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.
协同结合机制是生物学中的一个常见特征，它使各种各样基于蛋白质的分子机器能够调节从氧气摄取到细胞膜运输等一系列活动。然而，对于这种协同结合机制，我们还有很多不了解的地方，包括这些事件通常是如何增加此类蛋白质系统的整体稳定性的。测量这种协同稳定事件具有挑战性，因为它们需要在潜在化学计量比的混合物中分离和解析单个蛋白质复合物的结合状态，以便单独评估蛋白质的稳定性。在这里，我们报告了伴刀豆球蛋白A四聚体与一系列多糖配体结合的离子淌度-质谱结果。我们使用碰撞诱导解折叠，这是一种相对较新的方法，其作用类似于溶液中量热实验的气相模拟，来单独评估伴刀豆球蛋白A结合状态的稳定性。通过比较展开不同伴刀豆球蛋白A-配体化学计量比所需的激活电压差异，我们发现有证据表明，在结合大多数碳水化合物配体时，伴刀豆球蛋白A会发生协同稳定作用。我们通过评估广泛的配体、评估多种蛋白质电荷状态的解折叠特性，以及将我们的气相结果与溶液中量热实验获得的结果进行比较，来严格评估这一观察结果。
