Nakao Kaori, Steinhauser Alexandra, Durand Grégory, Soulié Marine, Rechberger Gerald N, Züllig Thomas, Keller Sandro, Tych Katarzyna
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Institute of Molecular Biosciences (IMB), NAWI Graz, University of Graz, Humboldtstr. 50, 8010 Graz, Austria.
Soft Matter. 2025 Aug 20;21(33):6596-6602. doi: 10.1039/d5sm00327j.
Nanodiscs have emerged as a powerful tool for studying membrane proteins in a lipid bilayer, with the standard approach relying on MSP-based nanodiscs that use detergent-mediated lipid exchange and encapsulation by MSP rings. However, this method may introduce artefacts from MSP interactions with the target protein and the nanodiscs constrained size. Here, we compare MSP-based nanodiscs with an alternative system using the amphiphile dodecyl-diglucoside (DDDG), which directly extracts membrane proteins along with their surrounding lipids from the cell membrane. Using a glutamate transporter homolog (GltTk) from as a model, we assessed the efficiency of extraction and purification, thermal stability, and substrate binding capacity of GltTk in each of the two nanodisc systems. Our findings demonstrate that DDDG-based nanodiscs are comparable to MSP-based nanodiscs but may provide greater conformational flexibility and avoid possible artefacts due to MSP-GltTk interactions. Consequently, they provide a competent alternative to MSP-based nanodiscs through direct extraction, thereby preserving the proteins native lipid environment. Both approaches support structural and functional studies, but their suitability depends on the specific application. MSP-based nanodiscs remain advantageous for studies requiring well-defined lipid compositions, while DDDG nanodiscs offer distinct advantages for investigating proteins where native lipids and conformational freedom are critical.
纳米圆盘已成为研究脂质双分子层中膜蛋白的有力工具，标准方法依赖于基于MSP的纳米圆盘，该方法利用去污剂介导的脂质交换并通过MSP环进行包封。然而，这种方法可能会引入MSP与靶蛋白相互作用以及纳米圆盘尺寸受限所产生的假象。在这里，我们将基于MSP的纳米圆盘与使用两亲性十二烷基二葡糖苷（DDDG）的替代系统进行比较，该系统直接从细胞膜中提取膜蛋白及其周围的脂质。以来自[具体来源未提及]的谷氨酸转运体同源物（GltTk）为模型，我们评估了两种纳米圆盘系统中GltTk的提取和纯化效率、热稳定性以及底物结合能力。我们的研究结果表明，基于DDDG的纳米圆盘与基于MSP的纳米圆盘相当，但可能提供更大的构象灵活性，并避免由于MSP - GltTk相互作用而产生的可能假象。因此，它们通过直接提取为基于MSP的纳米圆盘提供了一种有效的替代方案，从而保留了蛋白质的天然脂质环境。这两种方法都支持结构和功能研究，但它们的适用性取决于具体应用。基于MSP的纳米圆盘对于需要明确脂质组成的研究仍然具有优势，而DDDG纳米圆盘对于研究天然脂质和构象自由度至关重要的蛋白质具有明显优势。
