School of Chemistry and Physics, Pietermaritzburg Campus, University of KwaZulu-Natal, Private Bag X01, Scottsville, 3209, South Africa.
EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, EH9 3FJ, UK.
Rapid Commun Mass Spectrom. 2020 Jan 30;34(2):e8570. doi: 10.1002/rcm.8570.
A key element of studies that utilise ion mobility mass spectrometry (IM-MS) under native electrospray conditions for the analysis of protein-ligand binding is the maintenance of the native conformation of a protein during the removal of bulk solvent. Ruotolo and co-workers have demonstrated that the binding and subsequent dissociation of the anionic component of inorganic salts stabilise native protein conformations in the gas phase. In this study, we investigated the effect that organic acid fragments identified from a fragment-based drug discovery (FBDD) campaign might have on the gas-phase stability of carbonic anhydrase II (CA II).
We utilised native IM-MS to monitor changes in the conformation of CA II in the absence and presence of four acidic fragments. By performing a series of collision-induced unfolding (CIU) experiments we determined the effect of fragment binding on the gas-phase stability of CA II.
Binding and dissociation of acidic fragments result in increased gas-phase stability of CA II. CFU experiments revealed that the native-like compact gas-phase conformation of the protein is stable with higher degree of pre-activation when bound to a series of acidic fragments. Importantly, although acetate was present in high concentrations, the stabilising effect was not observed without the addition of the acidic fragments.
Binding and subsequent dissociation of acidic fragments from CA II significantly delayed CIU in a manner which is probably analogous to the effect of inorganic anions. Furthermore, we saw a slightly altered stabilising effect between the different fragments investigated in this study. This suggests that the prevention of CIU by organic acids may be tuneable to specific properties of a bound ligand. These observations may open avenues to exploit IM-MS as a screening platform in FBDD.
在保留蛋白质天然构象的情况下，利用电喷雾条件下的离子淌度质谱（IM-MS）分析蛋白质-配体结合的研究中，一个关键要素是在去除大量溶剂的过程中保持蛋白质的天然构象。Ruotolo 及其同事已经证明，无机盐的阴离子部分的结合和随后的解离稳定了气相中天然蛋白质构象。在这项研究中，我们研究了从基于片段的药物发现（FBDD）研究中鉴定出的有机酸片段可能对碳酸酐酶 II（CA II）的气相稳定性产生的影响。
我们利用天然 IM-MS 监测 CA II 在不存在和存在四种酸性片段的情况下构象的变化。通过进行一系列的碰撞诱导解折叠（CIU）实验，我们确定了片段结合对 CA II 气相稳定性的影响。
酸性片段的结合和解离导致 CA II 的气相稳定性增加。CFU 实验表明，当与一系列酸性片段结合时，蛋白质的天然样紧凑气相构象具有更高的预激活稳定性。重要的是，尽管醋酸盐的浓度很高，但如果没有添加酸性片段，则不会观察到稳定作用。
CA II 上酸性片段的结合和解离显著延迟了 CIU，其方式可能类似于无机阴离子的作用。此外，我们在这项研究中研究的不同片段之间观察到略微改变的稳定作用。这表明有机酸通过防止 CIU 可能是可以针对结合配体的特定性质进行调整的。这些观察结果可能为将 IM-MS 作为 FBDD 的筛选平台开辟途径。
