Kumar Smriti, Stover Lauren, Wang Lie, Bahramimoghaddam Hanieh, Zhou Ming, Russell David H, Laganowsky Arthur
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, United States.
bioRxiv. 2024 Jun 27:2024.06.27.601044. doi: 10.1101/2024.06.27.601044.
Native mass spectrometry (MS) is revealing the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer; however, commonly used detergents for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility for characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable characterization of lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find protein-lipid interactions are not only protein-dependent but can also be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl--dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery, as well as biochemical and structural investigations.
原生质谱（MS）正在揭示特定脂质在调节膜蛋白结构和功能方面的作用。溶解在去污剂中的膜蛋白通常会被引入质谱仪；然而，用于结构研究的常用去污剂，如十二烷基麦芽糖苷，往往会产生高电荷离子，导致蛋白质展开，从而降低其用于表征蛋白质-脂质相互作用的效用。因此，迫切需要开发方法来研究不同去污剂中的蛋白质-脂质相互作用。在这里，我们展示了诸如精胺和三甲胺-N-氧化物等电荷减少分子如何能够在不同的去污剂环境中表征脂质与细菌水通道（AqpZ）和氨通道（AmtB）与调节蛋白GlnK复合物的结合。我们发现蛋白质-脂质相互作用不仅取决于蛋白质，还会受到去污剂和电荷减少分子类型的影响。AqpZ与脂质的相互作用在LDAO（n-十二烷基-N,N-二甲基胺-N-氧化物）中增强，而AmtB-GlnK与脂质的相互作用在不同去污剂之间相当。荧光脂质结合试验也显示了AqpZ与脂质相互作用对去污剂的依赖性，这与原生质谱的结果一致。综上所述，原生质谱将在建立最佳实验参数方面发挥关键作用，这些参数对于各种应用，如药物发现以及生化和结构研究将是非常宝贵的。
