Institute of Atomic and Molecular Sciences, Academia Sinica, 1, Sec. 4, Roosevelt Rd., Taipei, 10617, Taiwan.
Department of Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei, 10617, Taiwan.
Sci Rep. 2018 Sep 10;8(1):13501. doi: 10.1038/s41598-018-31925-1.
Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.
将膜蛋白整合到膜模拟系统中是生物物理研究和结构测定的必要过程。单分散脂质纳米盘已被发现是一种合适的工具，因为它们提供了类似于天然的双层脂质环境。最近，一种共价环化的纳米盘（cND）被发现，它由膜支架蛋白（MSP）以环状形式组装，而不是传统的线性形式。共价环化的纳米盘已被证明具有更好的稳定性，然而，关于膜蛋白的最佳整合策略，以及嵌入 cND 中的膜蛋白的物理化学性质，尚未得到研究。细菌视紫红质（bR）是一种七跨膜螺旋（7TM）膜蛋白，它在嗜盐古菌的紫色膜上形成由三聚体 bR 组成的二维晶体。本文报道了 bR 三聚体在其活性形式下可以直接从其天然紫色膜整合到 cND 中。此外，通过优化高氯化钠浓度下的组装条件，实现了天然紫色膜纳米盘（PMND）的均匀性和高产量。此外，天然 PMND 被证明具有在不同 pH 值范围内组装的能力，这表明其制备条件具有灵活性。然后发现天然 PMND 不仅保留了 bR 的三聚体结构和 PM 中的大部分天然脂质，而且还保持了 bR 的光循环功能。这表明可以直接从天然膜环境中提取 7TM 膜蛋白，并将其组装到 cND 中，同时保留蛋白质构象和脂质组成，具有很大的应用潜力。
