PDF(Pubmed)
Abstract：
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.
摘要：
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是具有挑战性的，因为脂质可以同时在不同的位点以不同的特异性结合膜蛋白。这里,我们使用单突变体和双突变体开发了一种天然质谱（MS）方法，以测量水通道蛋白Z（AqpZ）上特定残基对心磷脂（CL）结合的相对能量贡献。我们首先在AqpZ上突变了潜在的脂质结合残基，和混合的突变和野生型蛋白与CL。通过使用天然MS在单一光谱中同时解析与突变体和野生型蛋白的脂质结合，我们直接确定了CL结合的相对亲和力，从而揭示了由突变引起的脂质结合的相对吉布斯自由能变化。比较不同的突变体显示，W14有助于最紧密的CL结合位点，R224有助于较低的亲和力位点。使用双突变循环，我们研究了W14和R224位点对CL结合的协同作用。总的来说,这种新颖的天然MS方法提供了对脂质与膜蛋白特异性位点结合的独特见解。
