Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm-1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

中文翻译：

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将 β 桶膜蛋白 OmpA 折叠成纳米圆盘

Nanodiscs (NDs) 是用于研究膜蛋白结构的小单层囊泡 (SUVs) 的绝佳替代品，但尚未表明膜蛋白能够自发折叠并插入自由扩散的 NDs 溶液中。在本文中，我们介绍了结合荧光、圆二色性和紫外共振拉曼光谱的 SDS-PAGE 差异迁移率研究，以确认外膜蛋白 A (OmpA) 自发折叠成预制 ND。NDs 中折叠的 OmpA 与 Arg-C 蛋白酶一起孵育，导致 OmpA 被消化成膜保护片段，其表观分子量为~26 kDa（主要成分）和~24 kDa（次要成分）。OmpA 折叠率在 ND 和 SUV 中均大于 88%。NDs 上吸附的 OmpA 中间体可以在低温下分离，并通过温度升高诱导折叠，类似于 SUV 的温度跳跃实验。OmpA 在 NDs 和 SUVs 中的圆二色光谱相似，表明为 β-桶二级结构。紫外共振拉曼光谱提供了 OmpA 折叠成 ND 的进一步证据，该光谱揭示了 ND 中折叠 OmpA 的强烈 785 cm-1 结构标记。NDs 和 SUVs 折叠的主要区别在于动力学；与 SUV 相比，ND 的折叠速度慢了两到三倍，这种降低的速度可以暂时归因于 ND 的特性。这些数据表明，NDs 可能是 SUVs 折叠实验的绝佳替代品，并提供光学透明度、样品均匀性、