Department of Biological Chemistry and Molecular Pharmacology , Harvard Medical School , Boston , Massachusetts 02115 , United States.
State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science , Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences , Shanghai 201203 , China.
Biochemistry. 2019 Sep 17;58(37):3834-3837. doi: 10.1021/acs.biochem.9b00636. Epub 2019 Sep 3.
The p7 protein encoded by the hepatitis C virus forms a cation-selective viroporin in the membrane. One of the most intriguing findings about the p7 viroporin is its unique hexameric structure in dodecylphosphocholine (DPC) micelles determined by nuclear magnetic resonance (NMR), but the hexameric structure was recently challenged by another NMR study of p7, also in DPC detergent, which claimed that the p7 in this detergent is monomeric. Here, we show that p7 oligomerization is highly sensitive to the detergent:protein ratio used in protein reconstitution and that the 40-fold difference in this ratio between the two studies was the cause of their different conclusions. In addition, we have performed extensive measurements of interchain paramagnetic relaxation enhancements (PREs) for p7 hexamers reconstituted in DPC micelles and in 1,2-dimyristoyl--glycero-3-phosphocholine/1,2-dihexanoyl--glycero-3-phosphocholine bicelles. In both cases, interchain PREs are overall consistent with the hexameric structure determined in micelles. Our data validate the overall architecture of the p7 hexamer while highlighting the importance of the detergent:protein ratio in membrane protein sample preparation.
丙型肝炎病毒编码的 p7 蛋白在膜中形成阳离子选择性的病毒孔道。关于 p7 病毒孔道最有趣的发现之一是，其在十二烷基磷酸胆碱 (DPC) 胶束中通过核磁共振 (NMR) 确定的独特六聚体结构，但最近另一项关于 DPC 洗涤剂中 p7 的 NMR 研究对其六聚体结构提出了质疑，该研究声称该洗涤剂中的 p7 是单体。在这里，我们表明 p7 寡聚化对用于蛋白质重组的洗涤剂与蛋白质的比例高度敏感，并且这两个研究之间的比例相差 40 倍是导致它们得出不同结论的原因。此外，我们还对在 DPC 胶束中和 1,2-二月桂酰基 - 甘油 -3-磷酸胆碱/1,2-二己酰基 - 甘油 -3-磷酸胆碱双胶束中重组的 p7 六聚体进行了广泛的链间顺磁弛豫增强 (PRE) 测量。在这两种情况下，链间 PRE 总体上与胶束中确定的六聚体结构一致。我们的数据验证了 p7 六聚体的整体结构，同时强调了在膜蛋白样品制备中洗涤剂与蛋白质的比例的重要性。
