Bavarian NMR Center at the Department of Chemistry, Technical University of Munich, Ernst-Otto-Fischer Strasse 2, 85748 Garching, Germany.
Bavarian NMR Center at the Department of Chemistry, Technical University of Munich, Ernst-Otto-Fischer Strasse 2, 85748 Garching, Germany; Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.
J Mol Biol. 2022 Dec 30;434(24):167861. doi: 10.1016/j.jmb.2022.167861. Epub 2022 Oct 20.
Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.
膜模拟物对于膜蛋白的结构和功能研究至关重要。一种有前途的基于脂质的系统是磷脂纳米盘，其中两个所谓的膜支架蛋白 (MSP) 的副本包裹在脂质双层片上。因此，纳米盘的大小取决于 MSP 的长度。此外，已经报道了共价 MSP 环化可以提高纳米盘的稳定性。然而，迄今为止，还没有对用于高分辨率 NMR 的环化和线性 MSP 纳米盘的生物物理性质进行更详细的比较分析。在这里，我们使用大量分析方法分析了环化和线性纳米盘的膜流动性和温度依赖性尺寸可变性。我们表明 MSP 环化不会改变纳米盘中的膜流动性。此外，我们表明环化版本的相变温度升高，而协同性降低。我们证明环化纳米盘在很大的温度范围内保持恒定的尺寸，与它们的线性 MSP 对应物相反。由于这种尺寸稳定性，环化纳米盘有利于在高温下进行膜蛋白的高分辨率 NMR 研究。尽管与线性纳米盘相比，它们的尺寸略大，但与类似尺寸的线性纳米盘中掺入的 VDAC1 相比，环化纳米盘中电压依赖性阴离子通道 1 (VDAC1) 的 3D NMR 实验具有明显改善的光谱质量。这项研究提供了证据，表明环化的 MSP 纳米盘是一种很有前途的工具，可以促进在天然脂质环境中对更大和更具挑战性的膜蛋白进行高分辨率 NMR 研究。
