Influenza A Virus M1 Oligomerization Figure 3. The oligomerization of M1N analyzed by gel filtration. (A) Purified M1N from nickel affinity chromatography yielded five peaks on Superdex 200. The volumes of elution were indicated. The samples from each peak were collected and analyzed by SDS-PAGE. (B) The 17.6-ml fraction (from panel A) was collected, concentrated and re-applied to the column at different concentrations, at pH 7.4. (C) Different concentrations of M1N all eluted as a single peak on the gel filtration column at pH 5.0. (D) Peak 1, 2, 3 correspond to the 9.8-ml, 11.3-ml, and 13.5-ml fractions of M1N re-applied on the gel filtration column at pH 7.4, respectively. Peak 4 corresponds to the 11.3-ml fraction re-applied on gel filtration column at pH 5.0. doi:10.1371/journal.pone.0037786.g003 25 kDa (Fig. S2), which is between two and three times the theoretical value of monomer (9.7 kDa). Consistent with the gel filtration result, the 17.3-ml fraction existed as one species with the diameter of 2.6 nm in dynamic light scatting (DLS) (Fig. S4). No other oligomers were formed, indicating that the 17.3-ml fraction is stable and that proteins of this fraction do not interact with each other. Sedimentation velocity analytical ultracentrifugation was also used to analyze the oligomerization states of C-terminal domain from the accurate molecular mass of protein species. M1C existed as a mono-disperse species with a sedimentation velocity value of 2.5 S at pH 7.4 (Fig. 6C). This c(s) value corresponds to a molecular mass of 19.6 kDa (Fig. 6D), indicating that M1C exists as a stable dimer in solution. The dimeric form of M1C was further confirmed by cross linking with glutaraldehyde, which resulted in the 17.3-ml fraction forming two bands on Tricine- SDS-PAGE. One band was at the same position as the untreated sample, and the other ran to the position of about 20 kDa, which is about the molecular mass of an M1C dimer (Fig. 6E). The molecular mass of M1C obtained by analytical gel filtration is larger than that obtained by analytical ultracentrifugation, possible due to its molecular shape. Together, these results indicated that there is direct interaction between C-terminal domains, and the C- terminal domain exists in dimeric form. In the GST pull down assay, no M1C was detected bound to GST-M1 (Fig. 6F). Combined with the results of the pull down assay of N-terminal domain, the data suggested that the N- terminal domain directly binds to the N-terminal domains of other M1 molecules, while the C-terminal domain forms a stable dimer that does not interact with other M1 molecules. As a result, we can conclude that the smallest oligomerization state of M1 is a dimer, and that dimeric M1 could form higher oligomers through direct interactions between N-terminal domains. To further analyze the structural characteristics of the C- terminal domain, several spectroscopy experiments were also performed. Far-UV CD spectra of M1C were collected over a PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e37786