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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)
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Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS)

机译：使用行波离子迁移率光谱法（TWIMS）研究Arixtra结合后抗凝血酶III的气相构象变化

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We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII-Arixtra complex. IM measurements of ATIII and ATIII-Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII-Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein-carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.
机译：我们使用具有良好特征的抗凝血酶III（ATIII）和具有肝素（Hp）活性的药物Arixtra，验证了离子淌度用于测量糖胺聚糖配体结合引起的蛋白质构象变化的实用性。肝素通过与丝氨酸蛋白酶抑制剂ATIII的相互作用已被用作治疗性抗凝药物数十年，它在凝血级联反应中起着核心作用。这种相互作用诱导ATIII内的构象变化，从而显着提高ATIII介导的抑制率。 Arixtra是最小的合成Hp，含有与ATIII结合所需的特定五糖序列。在这里，我们报告第一次行波离子迁移率质谱（TWIMS）研究ATIII与Arixtra相互作用引起的ATIII构象变化。天然电喷雾电离质谱技术可以平稳转移ATIII和ATIII-Arixtra配合物的天然拓扑。 IM对ATIII和ATIII-Arixtra配合物的测量显示出一个单一的结构，具有明确的碰撞截面（CCS）值。 ATIII与Arixtra相互作用的结果是，ATIII的CCS平均增加了3.6％，这与基于蛋白质晶体结构对CCS改变的理论估计非常吻合。在变性和非变性条件下对ATIII结合行为的比较证实了ATIII折叠三级结构对其生物学活性的重要性。结构类似于Arixtra但缺少中央葡糖胺残基上的3-O磺基的Hp寡糖显示出与ATIII的结合亲和力显着降低，但复合物的迁移行为没有变化，这与先前的研究表明3-O硫酸化影响与ATIII结合的平衡常数，但不影响相互作用方式。相比之下，Hp四糖的非特异性结合表现出更复杂的迁移行为，表明与ATIII的混杂相互作用。还评估了ATIII和ATIII-Arixtra复合物的碰撞活化作用，发现Arixtra的结合为ATIII提供了额外的抗折叠稳定性。总的来说，我们的结果验证了TWIMS保留蛋白质-碳水化合物复合物溶液结构的重要特征的能力，因此它可用于研究由糖胺聚糖配体的结合引起的蛋白质构象变化。

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