Investigation of Protein-Lipid Interactions Using Native Mass Spectrometry.
Investigation of Protein-Lipid Interactions Using Native Mass Spectrometry.
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DOI：
10.1007/978-1-0716-1585-0_3
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Laganowsky, Arthur
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文献类型：
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作者：
Cong, Xiao;Patrick, John W;Laganowsky, Arthur
整合膜蛋白嵌入生物膜中，其中各种脂质调节其结构和功能。迫切需要阐明这些脂质如何参与与膜蛋白功能障碍相关的生理和病理过程。天然质谱 (MS) 与离子迁移谱 (IM) 相结合，正在成为探测膜蛋白复合物及其与配体、脂质和其他小分子相互作用的强大工具。与其他生物物理方法不同，天然 IM-MS 可以解析单个配体/脂质结合事件。我们开发了一种使用天然 MS 与温度控制装置相结合的新方法，以确定单个配体或脂质与蛋白质结合事件的热力学参数。该方法已使用多种可溶性蛋白质配体系统进行了验证，其中将 MS 结果与传统生物物理技术（例如等温滴定量热法 (ITC) 和表面等离子共振 (SPR)）获得的结果进行比较。利用这些原理，可以阐明单个脂质与完整膜蛋白结合的热力学。在这里，我们使用大肠杆菌的氨通道（AmtB）作为模型膜蛋白。值得注意的是，观察到 AmtB 与具有不同头基和酰基链构型的脂质结合的不同热力学特征。此外，使用消除特定脂质结合位点的AmtB突变体，与野生型相比，在热力学特征中发现了明显的变化，这意味着这些特征可以识别参与特定脂质结合的关键残基，并可能区分特定脂质结合位点。本章提供了与温控天然 MS 相关的程序和发现，作为一种询问膜蛋白及其与脂质和其他分子相互作用的新方法。
Integral membrane proteins are embedded in biological membranes where various lipids modulate their structure and function. There exists a critical need to elucidate how these lipids participate in the physiological and pathological processes associated with the membrane protein dysfunction. Native mass spectrometry (MS), combined with ion mobility spectrometry (IM), is emerging as a powerful tool to probe membrane protein complexes and their interactions with ligands, lipids, and other small molecules. Unlike other biophysical approaches, native IM-MS can resolve individual ligand/lipid binding events. We have developed a novel method using native MS, coupled with a temperature-control apparatus, to determine the thermodynamic parameters of individual ligand or lipid binding events to proteins. This approach has been validated using several soluble protein-ligand systems wherein MS results are compared with those acquired from conventional biophysical techniques, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Using these principles, it is possible to elucidate the thermodynamics of individual lipid binding to integral membrane proteins. Herein, we use the ammonia channel (AmtB) from Escherichia coli as a model membrane protein. Remarkably, distinct thermodynamic signatures for AmtB binding to lipids with different headgroups and acyl chain configurations are observed. Additionally, using a mutant form of AmtB that abolishes a specific lipid binding site, distinct changes have been discovered in the thermodynamic signatures compared with the wild-type, implying that these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. This chapter provides procedures and findings associated with temperature-controlled native MS as a novel approach to interrogate membrane proteins and their interactions with lipids and other molecules.