Influenza A Virus M1 Oligomerization Figure 2. Cross-linking assay of M1 and GST pull-down assay detected the direct interaction between M1 molecules. (A) Lane 1 is M1 in the absence of cross-linker, and lane 2 is the cross-linked M1 dimer. (B) Glutathione-Sepharose bound GST-M1 or GST was incubated with hisM1 in the binding buffer (pH 7.4). After washing extensively with the buffer of different pH (7.4 or 5.0), the hisM1 bound to the beads was extracted and analyzed by immunoblotting with anti-His antibody. doi:10.1371/journal.pone.0037786.g002 Purified M1N protein from affinity chromatography was exchanged into a buffer of pH 5.0. The protein at the final concentration of 1.5 mg/ml was loaded onto the gel filtration column at pH 5.0. Only one fraction of M1N, which eluted at 17.4 ml, was observed (Fig. S3B). This fraction was collected and concentrated to higher concentration, yet it was still eluted with a symmetric profile at 17.4 ml (Fig. 3C). Like the intact protein, the N-terminal domain purified in acidic pH did not oligomerize after pH neutralization. Only the 17.4-ml fraction appeared on the gel filtration column after the sample buffer was neutralized to pH 7.4 (Fig. S3D). We also individually collected the 9.8-ml, 11.3-ml, and 13.5-ml fractions from the column at pH 7.4, and transferred them into a buffer of pH 5.0. Each sample was loaded on the gel filtration column at pH 5.0, and all the samples eluted at 17.4 ml. The 17.4-ml fraction generated from acidification of 11.3-ml fraction was shown in Fig. 3D. These results indicated that the interactions between M1N were disrupted in an acidic environ- ment, and moreover, demonstrated that the N-terminal domain contributes to the pH-dependent oligomerization characteristic of the intact protein. To more accurately observe the oligomerization status of M1N, we also performed sedimentation velocity analytical ultracentrifu- gation. Unfortunately, due to degradation issues, we could not resolve the sedimentation coefficient distributions (c(s)) of intact M1. The M1N samples were collected from a 17.6-ml fraction at pH 7.4 and a 17.4-ml fraction at pH 5.0 from the gel filtration column, and then concentrated to 1.2 mg/ml, respectively. The c(s) value was analyzed accordingly (see materials and methods). Sedimentation velocity of M1N at pH 5.0 gave rise to a single peak at 1.8 S (Fig. 4C). This corresponds to a molecular mass of 18.8 kDa (Fig. 4D), which closely agrees with the mass of its monomeric form. At pH 7.4, the data showed the presence of three species. The major species was at 1.8 S, and the sedimentation coefficients of the other two species were 3.5 S and 5.6 S (Fig. 4A), corresponding to molecular masses of 48 kDa and 96 kDa (Fig. 4B). No species higher than 100 kDa were observed. The ascending curve, which appeared at 15.0 S species, indicated that some of the sample might precipitate during centrifugation (Fig. 4A). Taken together, we concluded that the smallest oligomerization state of M1N is a monomer. M1N can form higher molecular mass oligomers at pH 7.4, but it exists only in monomeric form at pH 5.0. To test the self-interaction between N-terminal domains in solution, we examined their direct binding activity to a panel of GST fusion proteins. GST-M1 was used to pull down M1N as GST-M1N was expressed in insoluble form in E. coli. The possible interaction between M1N and M1C was excluded, as GST-M1 could not pull down M1C. M1N could bind to GST-M1 (Fig. 5A, lane 3), but not to GST alone at pH 7.4 (Fig. 5A, lane 2). The result suggested that N-terminal domains can directly interact with each other at neutral pH. The effect of pH on binding activity was further examined by incubating GST-beads-bound GST-M1 with M1N for 2 hours at pH 7.4, and then washing with acidic buffer (pH 5.0). No M1N was detected by anti-His antibody (Fig. 5A, lane 4). These data indicated that the direct interactions between N-terminal domain were disrupted by low pH and confirmed that oligomerization of N-terminal domain is pH-dependent. The C-terminal domain is a stable dimer The C-terminal domain (residues 165–252) of M1 was expressed as a GST-fusion protein. M1C was purified using Glutathione Sepharose 4B resin. After cleavage of the GST tag at the N-terminal by PreScissors protease, the protein migrated as a single band of about 10 kDa in the PAGE (Fig. S1A). We then analyzed the oligomerization of M1C with gel filtration. After the GST tag was removed, M1C was loaded on the gel filtration column at pH 7.4, yielding a single fraction with an elution volume of 17.3 ml (Fig. 6A). That fraction was collected, concentrated and re-applied on the column. Unlike the N- terminal domain, only one fraction of 17.3 ml eluted on the column, independent of pH and concentration variations (Fig. 6B). The apparent molecular mass of the species was calculated to be PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37786