<br>同时对单突变体和双突变体进行天然质谱分析以探测脂质与膜蛋白的结合,Analytical Chemistry

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同时对单突变体和双突变体进行天然质谱分析以探测脂质与膜蛋白的结合
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-06-10 , DOI: 10.1021/acs.analchem.4c01704
Hiruni S Jayasekera ₁ , Farhana Afrin Mohona ₁ , Megan Ewbank ₁ , Michael T Marty ₁

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脂质是膜蛋白结构和功能的关键调节剂。然而，研究蛋白质-脂质相互作用的热力学具有挑战性，因为脂质可以同时在不同位点以不同的特异性结合膜蛋白。在这里，我们开发了一种使用单突变体和双突变体的天然质谱 (MS) 方法来测量水通道蛋白 Z (AqpZ) 上特定残基对心磷脂 (CL) 结合的相对能量贡献。我们首先突变了 AqpZ 上潜在的脂质结合残基，并将突变型和野生型蛋白与 CL 混合在一起。通过使用天然 MS 在单一光谱中同时解析脂质与突变型和野生型蛋白质的结合，我们直接确定了 CL 结合的相对亲和力，从而揭示了突变引起的脂质结合的相对吉布斯自由能变化。比较不同的突变体表明，W14 有助于形成最紧密的 CL 结合位点，而 R224 有助于形成较低的亲和力位点。使用双突变体循环，我们研究了 W14 和 R224 位点对 CL 结合的协同作用。总体而言，这种新颖的天然 MS 方法为脂质与膜蛋白特定位点的结合提供了独特的见解。

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Simultaneous Native Mass Spectrometry Analysis of Single and Double Mutants To Probe Lipid Binding to Membrane Proteins

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein–lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.

更新日期：2024-06-10

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