Structural Biophysics, Niels Bohr Institute , University of Copenhagen , Universitetsparken 5 , 2100 Copenhagen , Denmark.
Langmuir. 2018 Oct 23;34(42):12569-12582. doi: 10.1021/acs.langmuir.8b01503. Epub 2018 Oct 10.
Phospholipid nanodiscs have quickly become a widely used platform for studies of membrane proteins. However, the molecular self-assembly process that ultimately should place a membrane protein inside a nanodisc is not well understood. This poses a challenge for a successful high-yield reconstitution of general membrane proteins into nanodiscs. In the present work, the self-assembly process of POPC-MSP1D1 nanodiscs was carefully investigated by systematically modulating the reconstitution parameters and probing the effect with a small-angle X-ray scattering analysis of the resulting nanodiscs. First, it was established that nanodiscs prepared using the standard protocol followed a narrow but significant size distribution and that the formed nanodiscs were stable at room temperature over a time range of about a week. Systematic variation of the POPC/MSP1D1 stoichiometry of the reconstitution mixture showed that a ratio of less than 75:1 resulted in lipid-poor nanodiscs, whereas ratios of 75:1 and larger resulted in nanodiscs with constant POPC/MSP1D1 ratios of 60:1. A central step in the self-assembly process consists in adding detergent-absorbing resin beads to the reconstitution mixture to remove the reconstitution detergent. Surprisingly, it was found that this step did not play a significant role for the shape and stoichiometry of the formed nanodiscs. Finally, the effect of the choice of detergent used in the reconstitution process was investigated. It was found that detergent type is a central determining factor for the shape and stoichiometry of the formed nanodiscs. A significantly increasing POPC/MSP1D1 stoichiometry of the formed nanodiscs was observed as the reconstitution detergent type is changed in the order: Tween80, DDM, Triton X-100, OG, CHAPS, Tween20, and Cholate, but with no simple correlation to the characteristics of the detergent. This emphasizes that the detergents optimal for solution storage and crystallization of membrane proteins, in particular DDM, should not be used alone for nanodisc reconstitution. However, our data also show that when applying mixtures of the reconstitution detergent cholate and the storage detergents DDM or OG, cholate dominates the reconstitution process and nanodiscs are obtained, which resemble those formed without storage detergents.
磷脂纳米盘已迅速成为研究膜蛋白的广泛应用平台。然而，将膜蛋白置于纳米盘中的分子自组装过程尚不清楚。这对于将一般的膜蛋白成功高效地重组到纳米盘中是一个挑战。在本工作中，通过系统地调节重组参数，并通过小角度 X 射线散射分析来探测所得纳米盘的效果，仔细研究了 POPC-MSP1D1 纳米盘的自组装过程。首先，已经确定使用标准方案制备的纳米盘遵循狭窄但显著的尺寸分布，并且在室温下形成的纳米盘在大约一周的时间范围内是稳定的。系统地改变重组混合物中 POPC/MSP1D1 的比例表明，小于 75:1 的比例导致脂质不足的纳米盘，而比例为 75:1 及更大的比例导致纳米盘的 POPC/MSP1D1 比例保持不变，为 60:1。自组装过程中的一个关键步骤是将去污剂吸收树脂珠添加到重组混合物中以去除重组去污剂。令人惊讶的是，发现该步骤对于形成的纳米盘的形状和比例没有显著影响。最后，研究了在重组过程中使用的去污剂类型的影响。发现去污剂类型是形成的纳米盘的形状和比例的决定性因素。随着重组去污剂类型按 Tween80、DDM、Triton X-100、OG、CHAPS、Tween20 和胆酸钠的顺序变化，观察到形成的纳米盘的 POPC/MSP1D1 比例显著增加，但与去污剂的特性没有简单的相关性。这强调了用于膜蛋白溶液储存和结晶的最佳去污剂，特别是 DDM，不应单独用于纳米盘重组。然而，我们的数据还表明，当应用重组去污剂胆酸钠和储存去污剂 DDM 或 OG 的混合物时，胆酸钠主导重组过程，得到类似于没有储存去污剂形成的纳米盘。
