Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
J Phys Chem B. 2017 Jun 1;121(21):5312-5321. doi: 10.1021/acs.jpcb.7b01705. Epub 2017 May 23.
EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (T) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the T titration experiment indicated an increase in T values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.
EPR 光谱研究膜蛋白在生理相关的天然膜结合状态下极具挑战性，这是由于在不均匀的样品制备和自旋标记的动态运动中观察到的复杂性。传统上，由于其较小的尺寸和均一性，胶束去污剂是最广泛用于膜蛋白的膜类似物，可通过溶液 NMR 光谱提供高分辨率结构分析。然而，通常很难检查在胶束环境中的蛋白质结构是否与相应的膜结合状态相同。最近，脂质体纳米颗粒已被引入作为用于膜蛋白结构研究的潜在良好的膜模拟系统。然而，对于结合到脂质体纳米颗粒中的自旋标记膜蛋白的详细表征仍然缺乏。在这项工作中，脂质体纳米颗粒被用作用于通过基于位置的自旋标记 EPR 光谱研究整体膜蛋白 KCNE1 的结构和动态特性的膜模拟系统。通过 CW-EPR 滴定实验进行的自旋标记的 KCNE1 与脂质体纳米颗粒结合的特征在于 EPR 光谱线形状分析的 CW-EPR 滴定实验和相记忆时间 (T) 测量的脉冲 EPR 滴定实验。CW-EPR 滴定实验表明，随着 SMA 聚合物的加入，谱线展宽增加，当脂质与聚合物的重量比为 1:1 时，接近刚性极限，这提供了蛋白质-脂质复合物的明显溶解。同样，T 滴定实验表明，随着 SMA 聚合物的加入，T 值增加，当脂质与聚合物的重量比为 1:2 时，T 值接近 2 μs。此外，在脂质体纳米颗粒中的 KCNE1 的六个内部和六个外部膜自旋标记探针上进行了 CW-EPR 光谱线形状分析。与脂质体囊泡样品相比，对于脂质体纳米颗粒样品，结果表明位于膜内和膜外的 KCNE1 的自旋标记残基的 EPR 光谱线展宽和相应的反向中心线宽有显著差异。这些结果与 KCNE1 的溶液 NMR 结构一致。这项研究将有助于研究膜蛋白的结构和动态特性的研究人员。
