Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

中文翻译：

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用于 NMR 研究的肽纳米盘中 19F 标记的细胞色素 b5 的表达、纯化和功能重建。


微粒体细胞色素 b5 (cytb5) 是一种膜结合蛋白，能够在 cytP450s 单加氧酶反应中向细胞色素 P450s (cytP450s) 提供第二个电子。最近的研究证明了 cytb5 跨膜结构域通过稳定其单体结构在与 cytP450 相互作用中的重要性。虽然最近的 NMR 研究为膜环境中 ~16-kDa cytb5 和 ~57-kDa cytP450 可溶性结构域之间的结构相互作用提供了高分辨率的见解，但还需要研究来探测跨膜区域中的残基以获得分子间距离约束，以更好地理解近天然膜环境中非常大尺寸的 cytb5-cytP450 复合物结构。在这项研究中，我们报告了 19F 标记的全长兔 cytb5 在基于肽的纳米盘中的表达、纯化和功能重建，以使用 NMR 光谱进行结构研究。尺寸排阻色谱、动态光散射、透射电子显微镜和核磁共振实验表明 cytb5 在 4F 肽基脂质纳米盘中稳定重建。报告的结果表明，使用 19F NMR 实验来研究在肽纳米盘中重构的 19F 标记（使用 5-氟色氨酸 (5FW)）cytb5，并通过溶液 19F NMR 实验检测跨膜结构域的残基。还提供了揭示 cytb5 跨膜结构域与全长兔细胞色素 P450 2B4 (CYP2B4) 相互作用的 19F NMR 结果。 我们期望本研究中提出的结果有助于设计方法来探测膜蛋白跨膜结构域的结构、动力学和功能作用，并测量分子间 19F-19F 距离约束以确定跨膜结构域之间的结构相互作用。