Sedimentation of eE2-C656S at pH 7 indicates the presence of two dominant species, a monomeric form (60 to 70 kDa) and a dimeric form (80 to 130 kDa) (Fig. 5A and B). There is a minor third species that has the approximate molecular mass of a trimer (150 to 200 kDa). The relative proportions kinase inhibitor Wortmannin of the three species are 65%:29%:6% (monomer:dimer:trimer). Since analytical ultracentrifugation failed to detect large aggregates, we assume that the trimer corresponds to the small peak that eluted in the void volume of SEC. The monomer has a wide distribution of frictional ratio in the range of 1.0 to 2.0, where a perfect sphere would have a frictional ratio of 1.0. It is interesting to note that the frictional ratio shows a decreasing trend with increasing molecular mass, suggesting a more globular shape for the oligomeric forms.

To determine if the ratio of oligomers or protein globularity was dependent on the amount of protein measured, the analysis was performed at two different concentrations (Fig. (Fig.5).5). There is no appreciable difference in the molecular mass, ratio of the different species, or shape distributions at the two concentrations used in this analysis (OD230s of 0.25 and 0.8), indicating little or no effect due to mass action at these concentrations. FIG. 5. Analytical ultracentrifugation data for eE2-C656S at pH 7. Two-dimensional spectrum/Monte Carlo analysis of HCN sedimentation velocity data. Measurements of eE2 were made at a low concentration (0.25 OD230) (A) or at a higher concentration (0.8 OD230 …

The HCV glycoproteins are predicted to be class II fusion proteins due to their relatedness to proteins of alphaviruses and flaviviruses. Class II fusion proteins are composed of mostly ��-sheet structure and do not undergo major rearrangements in secondary structure upon exposure to low pH (34). CD was employed to determine the secondary structure of eE2 and whether there are any changes upon lowering of the pH. CD spectra of both eE2 and eE2-C656S measured at pH 7.0 exhibited a minimum at about 203 nm (Fig. 6A and B). Multilinear regression analysis (data not shown) suggested that the eE2 spectrum is consistent with a protein composed of predominantly ��-sheet and random coil secondary structure with little to no alpha-helical content. The CD spectra measured at pH 5 are superimposable on those measured at pH 7 for both eE2 and eE2-C656S.

From these data, we conclude that eE2 has mostly ��-sheet and random coil structure and does not undergo major changes in secondary structure, similar to the flavivirus envelope GSK-3 protein. FIG. 6. CD spectroscopy of eE2 (A) or eE2-C656S (B) at pH 7 and pH 5. CD spectra are shown as millidegrees versus wavelength (nm). Error bars for each data point are given. J6 eE2 is recognized by antibodies from patients chronically infected with different genotypes of HCV. The presence of high levels of anti-E2 antibodies in HCV-infected human serum has been reported (3).