Critical Effect of the Detergent:Protein Ratio on the Formation of the Hepatitis C Virus p7 Channel

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-phosphocholinebicelles. 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.