Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA.
Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA.
Anal Biochem. 2022 Jun 15;647:114692. doi: 10.1016/j.ab.2022.114692. Epub 2022 Apr 21.
Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.
尽管膜蛋白在生物功能和生理学中起着重要作用，但由于缺乏仿生系统来保持蛋白质处于其天然膜环境中，因此研究它们仍然具有挑战性。冷冻电子显微镜 (Cryo-EM) 作为分析膜蛋白结构的强大工具正在兴起。然而，Cryo-EM 和其他膜蛋白分析最好在天然脂质双层中进行研究。尽管传统的仿生系统有其缺点，限制了它们在膜蛋白研究中的应用。作为替代方案，苯乙烯-马来酸共聚物用于与 POPC:POPG 脂质形成纳米颗粒。这些苯乙烯马来酸脂质纳米颗粒 (SMALPs) 的传统表征包括动态光散射 (DLS)、电子顺磁共振 (EPR)、核磁共振 (NMR) 和透射电子显微镜 (TEM)。在这项研究中，开发了一种新方法，该方法利用 TEM 网格上的苯乙烯-马来酸共聚物 (SMA) 薄膜作为底物的 SMALPs。通过直接将 POPC:POPG 脂质囊泡添加到涂有 SMA 的网格上，可以形成、可视化和通过 TEM 对 SMALPs 进行表征，而无需在成像前将其在溶液中制备。我们设想这些功能化的网格可以帮助进行单颗粒标本制备，提高结构生物学和生物物理技术（如 Cryo-EM）的效率。
