Kit Melanie Cheung See, Carvalho Veronica V, Vilseck Jonah Z, Webb Ian K
Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana 46202, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Int J Mass Spectrom. 2021 May;463. doi: 10.1016/j.ijms.2021.116549. Epub 2021 Feb 17.
Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.
蛋白质分子内的相互作用对于维持蛋白质三级结构以及理解蛋白质的功能至关重要。离子淌度-质谱联用技术（IM-MS）已成为结构生物学中广泛应用的一种方法，因为它能够快速测量碰撞截面（CCS），而碰撞截面可反映所研究生物分子的气相构象。气相离子/离子反应针对具有特定化学性质的氨基酸残基，通过质谱可鉴定修饰位点。在本研究中，将具有静电反应性的气相离子/离子化学与IM-MS相结合，以表征在水性和变性条件下电喷雾的泛素之间的结构变化。通过离子/离子反应使磺基-NHS乙酸酯与泛素发生静电附着，并通过电子捕获解离（ECD）进行碎片化，从而确定泛素中最易接近的质子化位点，因为磺酸根基团与可接近的质子化侧链形成静电复合物。从不同溶液条件下通过ECD鉴定出的质子化位点各不相同，在某些情况下，反映出诸如维持天然蛋白质结构的盐桥等相互作用的破坏。这与先前发表的文献一致，文献表明在低pH下高甲醇浓度会导致泛素的结构从天然（N）状态转变为更细长的A状态。使用气相静电交联试剂的结果也表明了类似的结构变化，并进一步证实了甲醇和酸在促进更展开构象方面的作用。由于交联试剂对两个反应位点有距离限制，这些数据对于指导分子动力学生成的计算结构很有价值。本文介绍的研究描述了一种有前景的策略，该策略可以检测目标氨基酸残基局部环境中的细微变化，从而了解整体蛋白质结构的变化。
