Scheyer Matthew W, Campbell Conner, William Patrick L, Hussain Mustakim, Begum Afsana, Fonseca Sebastian Escobar, Asare Isaac K, Dabney Peyton, Dabney-Smith Carole, Lorigan Gary A, Sahu Indra D
Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA.
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Biophys Chem. 2023 Oct;301:107080. doi: 10.1016/j.bpc.2023.107080. Epub 2023 Jul 26.
One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (T) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins.
膜蛋白增溶的主要挑战之一是为其生物物理研究找到最佳的生理环境。电子顺磁共振光谱（EPR光谱）是研究大分子结构和动力学特性的强大生物物理技术。然而，膜蛋白样品制备中的挑战以及自旋标记的灵活运动限制了EPR光谱在大多数处于生理膜结合状态的膜蛋白系统中的应用。最近，脂质盘纳米颗粒或苯乙烯-马来酸共聚物-脂质纳米颗粒（SMALPs）已成为用于研究膜蛋白结构的膜模拟系统。然而，其用于膜蛋白研究的详细表征仍知之甚少。最近，我们使用EPR光谱对重构到脂质盘纳米颗粒中的钾通道膜蛋白KCNQ1电压传感结构域（KCNQ1-VSD）和KCNE1进行了表征。在本研究中，将含有柔性N端和C端的钾通道辅助蛋白KCNE3封装到蛋白脂质体和脂质盘纳米颗粒中，并使用基于氮氧化物的定点自旋标记EPR光谱对其结构和动力学特性进行表征。连续波EPR线形分析数据表明，随着苯乙烯-马来酸（SMA）聚合物的加入，光谱线宽增加，接近刚性极限，从而使蛋白质-脂质复合物得到均匀稳定。同样，EPR双电子-电子共振（DEER）测量表明，与蛋白脂质体相比，将样品掺入脂质盘纳米颗粒后，随着相位记忆时间（T）值的增加，距离测量的质量得到提高。这些结果与KCNE3的溶液核磁共振结构以及脂质盘纳米颗粒中其他膜蛋白的EPR研究结果一致。这项研究以及我们早期的研究将提供参考表征数据，这将有助于膜蛋白研究人员研究具有挑战性的膜蛋白的结构动力学。
