Influenza A Virus M1 Oligomerization Figure 1. The oligomerization of M1 analyzed by gel filtration. The oligomerization of M1 was analyzed on a Superdex 200 HR 10/30 column. (A) Purified M1 from nickel affinity chromatography yielded four peaks, whose elution volumes are indicated. The samples from each peak were collected and analyzed by SDS-PAGE. (B) The 15.8-ml fraction (from panel A) was collected, concentrated and re-applied on the gel filtration column at different concentrations, at pH 7.4. (C) Different concentrations of M1 all eluted as a single peak on the gel filtration column at pH 5.0. (D) Peak 1, 2, 3 correspond to the 10.1-ml, 11.2-ml, and 13.5-ml fractions of M1 re-applied on the gel filtration column at pH 7.4, respectively. Peak 4 corresponds to the 11.2-ml fraction re-applied on the gel filtration column at pH 5.0. doi:10.1371/journal.pone.0037786.g001 The N-terminal domain is responsible for the pH- dependent oligomerization of M1 To determine the roles of the two domains in M1 oligomer- ization, we further analyzed the oligomerization behavior of the N-terminal (1–170 aa) and C-terminal (165–252 aa) domains. Recombinant M1N was constructed with a C-terminal His6-tag and expressed in soluble form in E. coli. M1N was purified by nickel affinity chromatography and showed a single band of about 18 kDa on Tricine-SDS-PAGE (Fig. S1A, lane 5). Far-UV CD spectroscopy showed that the secondary structure of the recom- binant protein was composed of 72% a-helical and 28% random coil, which was consistent with the crystal structure (70% a-helical) (Fig. S1B). M1N purified by affinity chromatography to 0.9 mg/ml was loaded onto the gel filtration column at pH 7.4. The sample yielded five fractions with the elution volumes of 9.8 ml, 11.3 ml, 13.5 ml, 16.0 ml, and 17.6 ml (Fig. 3A). The apparent molecular mass of the 17.6-ml fraction of M1N was estimated to be 19 kDa, which is consistent with monomeric M1N (19.8 kDa) (Fig. S2). Then the protein was concentrated to different concentrations and re-applied to the gel filtration column. Similar to the intact protein, M1N also exhibited a concentration-dependent oligomer- ization. Only a single 17.6-ml fraction was observed on the gel filtration column when the loaded protein concentration was 0.3 mg/ml. However, the 0.5 mg/ml sample produced five fractions. The elution volumes of the fractions were 9.8 ml, 11.3 ml, 13.5 ml, 16.0 ml, and 17.6 ml, corresponding to the molecular masses of 654 kDa, 436 kDa, 169 kDa, 51 kDa, and 19 kDa, respectively. The proportion of the first four oligomer- ization fractions also increased with increasing sample concentra- tion (Fig. 3B). When the loaded protein concentration was above 1.0 mg/ml, some of the protein precipitated after incubation at room temperature for 1 h. Also, we collected 9.8-ml, 11.3-ml, and 13.5-ml fractions individually and re-applied each sample to the gel filtration column. The elution volumes of the fractions stayed the same and no other oligomers were observed on the column at pH 7.4 (Fig. 3D), even when the concentrations of the 9.8-ml, 11.3-ml, and 13.5-ml fractions were diluted to lower than 0.1 mg/ ml (data not shown). The data clearly suggested that, like the intact M1, the N-terminal domain can form multi-order oligomers at neutral pH, and that the oligomerization process of M1N is also irreversible. PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37786