Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
J Phys Chem B. 2017 Oct 5;121(39):9185-9195. doi: 10.1021/acs.jpcb.7b07631. Epub 2017 Sep 26.
Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study structural and dynamic properties of membrane proteins. The most widely used spin label is methanthiosulfonate (MTSL). However, the flexibility of this spin label introduces greater uncertainties in EPR measurements obtained for determining structures, side-chain dynamics, and backbone motion of membrane protein systems. Recently, a newer bifunctional spin label (BSL), 3,4-bis(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome the dynamic limitations associated with the MTSL spin label and has been invaluable in determining protein backbone dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing more accurate information about the structure and dynamics of several proteins, a detailed characterization of the spin label is still lacking. In this study, we characterized BSLs by performing CW-EPR spectral line shape analysis as a function of temperature on spin-labeled sites inside and outside of the membrane for the integral membrane protein KCNE1 in POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles. The experimental data revealed a powder pattern spectral line shape for all of the KCNE1-BSL samples at 296 K, suggesting the motion of BSLs approaches the rigid limit regime for these series of samples. BSLs were further utilized to report for the first time the distance measurement between two BSLs attached on an integral membrane protein KCNE1 in POPC/POPG lipid bilayers at room temperature using dipolar line broadening CW-EPR spectroscopy. The CW dipolar line broadening EPR data revealed a 15 ± 2 Å distance between doubly attached BSLs on KCNE1 (53/57-63/67) which is consistent with molecular dynamics modeling and the solution NMR structure of KCNE1 which yielded a distance of 17 Å. This study demonstrates the utility of investigating the structural and dynamic properties of membrane proteins in physiologically relevant membrane mimetics using BSLs.
定点自旋标记（SDSL）与电子顺磁共振（EPR）光谱学相结合，是研究膜蛋白结构和动态特性的一种非常强大的技术。最广泛使用的自旋标记物是甲硫磺酸酯（MTSL）。然而，这种自旋标记物的灵活性在确定膜蛋白体系结构、侧链动力学和骨架运动的 EPR 测量中引入了更大的不确定性。最近，一种新型双功能自旋标记物（BSL），3,4-双（甲硫磺酸基甲基）-2,2,5,5-四甲基-2,5-二氢-1H-吡咯-1-氧基，已被引入以克服与 MTSL 自旋标记物相关的动态限制，并在确定蛋白质骨架动力学和残基间距离方面非常有价值，因为其内部运动受限且尺寸限制较少。虽然 BSL 在提供有关几种蛋白质的结构和动力学的更准确信息方面取得了成功，但对自旋标记物的详细特征描述仍然缺乏。在这项研究中，我们通过在膜内和膜外的自旋标记位点上进行连续波 EPR 谱线形状分析，以温度为函数，对整合膜蛋白 KCNE1 在 POPC/POPG 脂质双层和 POPC/POPG 脂质体中的 BSL 进行了表征。实验数据表明，在 296 K 时，所有 KCNE1-BSL 样品均呈现粉末状谱线形状，表明这些系列样品中 BSL 的运动接近刚性极限状态。BSL 进一步被用于首次报道在室温下使用双自旋标记物在整合膜蛋白 KCNE1 上的距离测量，该双自旋标记物位于 POPC/POPG 脂质双层中。连续波偶极线宽 EPR 数据显示，在 KCNE1（53/57-63/67）上双连接的 BSL 之间的距离为 15±2 Å，这与分子动力学模拟和 KCNE1 的溶液 NMR 结构一致，该结构得出的距离为 17 Å。这项研究表明，在生理相关的膜模拟物中使用 BSL 研究膜蛋白的结构和动态特性具有实用性。
