Critical Effect of the Detergent:Protein Ratio on the Formation of the Hepatitis C Virus p7 Channel.
Critical Effect of the Detergent:Protein Ratio on the Formation of the Hepatitis C Virus p7 Channel.
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去污剂：蛋白质比例对丙型肝炎病毒 p7 通道形成的关键影响。
DOI：
10.1021/acs.biochem.9b00636
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Chou,JamesJ
中科院分区：
文献类型：
--
作者：
Chen,Wen;OuYang,Bo;Chou,JamesJ
由丙型肝炎病毒编码的p7蛋白在膜中形成阳离子选择性病毒孔蛋白。关于p7病毒孔蛋白的最有趣的发现之一是通过核磁共振（NMR）确定的其在十二烷基磷酸胆碱（DPC）胶束中的独特的六聚体结构，但是六聚体结构最近受到另一项关于p7的NMR研究的挑战，该研究也在DPC洗涤剂中，该研究声称该洗涤剂中的p7是单体的。在这里，我们表明p7寡聚化对蛋白质重建中使用的去污剂：蛋白质比率高度敏感，并且两项研究之间该比率的40倍差异是导致其不同结论的原因。此外，我们已经进行了广泛的测量链间顺磁弛豫增强（PREs）的p7六聚体重构DPC胶束和1，2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱/1，2-二己酰-sn-甘油-3-磷酸胆碱bicelles。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p7六聚体的整体架构，同时强调了洗涤剂：蛋白质比例在膜蛋白样品制备中的重要性。
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-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-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.