Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry
Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry
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DOI：
10.1021/jacs.6b01771
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发表时间：
2016-04-06
影响因子：
15
通讯作者：
Laganowsky, Arthur
中科院分区：
文献类型：
--
作者：
Cong, Xiao;Liu, Yang;Laganowsky, Arthur
膜蛋白嵌入生物膜中，不同的化学性质的脂质环境可以调节其结构和功能。然而，控制分子识别和脂质与膜蛋白相互作用的热力学却知之甚少。在这里，我们报告了一种使用天然质谱（MS）的方法，以确定单个配体与蛋白质结合事件的热力学。与传统方法不同，天然质谱可以解析单个配体结合事件，并与控制温度的装置相结合，确定结合热力学参数，例如蛋白质脂质相互作用。我们使用三种可溶性蛋白质配体系统（麦芽糖结合蛋白、溶菌酶和氮调节蛋白）验证了我们的方法，并获得了与等温滴定量热法和表面等离子体共振相似的结果。我们还首次确定了单个脂质与氨通道（AmtB）结合的热力学，氨通道是大肠杆菌的一种完整膜蛋白。值得注意的是，我们观察到不同脂质结合的不同热力学特征以及可变链长结合脂质的熵焓补偿。此外，利用AmtB的突变形式，消除了特定的磷脂酰甘油（PG）结合位点，我们观察到结合PG的热力学特征的明显变化，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，并可能区分特定的脂质结合位点。
Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein lipid interactions. We validated our approach using three soluble protein ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.