Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase.
Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase.
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DOI：
10.1021/ac5037022
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发表时间：
2015-01-20
影响因子：
7.4
通讯作者：
Ashcroft AE
中科院分区：
文献类型：
--
作者：
Calabrese AN;Watkinson TG;Henderson PJ;Radford SE;Ashcroft AE
非共价质谱（MS）正在成为一种宝贵的技术来探测膜蛋白（MPs）的结构、相互作用和动力学。然而，使用洗涤剂溶解的蛋白质在气相中维持原生MP构象通常具有挑战性，并且可能限制结构分析。amphipol，如表征良好的A8-35，是一种替代试剂，能够保持MPs在无洗涤剂溶液中的溶解度。在这项工作中，A8-35保持MPs结构完整性的能力被电喷雾电离-离子迁移谱-质谱（ESI-IMS-MS）与常用的洗涤剂十二烷基麦糖苷进行了系统的比较。从两个主要结构类中选择MPs进行分析，包括两个β-桶状外MPs， PagP和OmpT（分别为20.2和33.5 kDa），两个α-螺旋蛋白，Mhp1和GalP（分别为54.6和51.7 kDa）。对观察到的离子的旋转平均碰撞截面的评估表明，洗涤剂溶解的MPs的天然结构并不总是保留在气相中，可以检测到塌陷和未折叠的物种。相比之下，通过ESI-IMS-MS分析所研究的双酚类溶解MPs的电荷态分布与较少的气相诱导展开一致，并且存在低电荷离子，其碰撞截面与高分辨率结构数据计算的结果相当。数据表明，A8-35比十二烷基麦芽糖更有效地维持天然MP的结构和在气相中的相互作用，允许非共价ESI-IMS-MS分析来自两种主要结构类的MP，而十二烷基麦芽糖胶束的气相解离导致显著的气相展开，特别是对于α-螺旋MPs。
Noncovalent mass spectrometry (MS) is emerging as an invaluable technique to probe the structure, interactions, and dynamics of membrane proteins (MPs). However, maintaining native-like MP conformations in the gas phase using detergent solubilized proteins is often challenging and may limit structural analysis. Amphipols, such as the well characterized A8-35, are alternative reagents able to maintain the solubility of MPs in detergent-free solution. In this work, the ability of A8-35 to retain the structural integrity of MPs for interrogation by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is compared systematically with the commonly used detergent dodecylmaltoside. MPs from the two major structural classes were selected for analysis, including two β-barrel outer MPs, PagP and OmpT (20.2 and 33.5 kDa, respectively), and two α-helical proteins, Mhp1 and GalP (54.6 and 51.7 kDa, respectively). Evaluation of the rotationally averaged collision cross sections of the observed ions revealed that the native structures of detergent solubilized MPs were not always retained in the gas phase, with both collapsed and unfolded species being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized MPs studied resulted in charge state distributions consistent with less gas phase induced unfolding, and the presence of lowly charged ions which exhibit collision cross sections comparable with those calculated from high resolution structural data. The data demonstrate that A8-35 can be more effective than dodecylmaltoside at maintaining native MP structure and interactions in the gas phase, permitting noncovalent ESI-IMS-MS analysis of MPs from the two major structural classes, while gas phase dissociation from dodecylmaltoside micelles leads to significant gas phase unfolding, especially for the α-helical MPs studied.