M1C as glutathione S-transferase (GST) fusion protein. Cells were grown at 37uC in LB medium containing ampicillin, 100 mg/ml; kanamycin, 50 mg/ml to an OD600 of 0.8–1.0. The culture was harvested by centrifugation after induction with 0.5 mM IPTG (isopropyl-1-thio-b-D-galactopyanoside) at 16uC for 11 h. Then the cells were resuspended in binding buffer (His6-tagged proteins: 20 mM Tris, 500 mM NaCl, 20 mM imidazole, 0.1 mM PMSF, pH 7.9; GST-tagged protein: Phosphate buffer saline (PBS), 0.1 mM PMSF, pH 7.4) and lysed by sonication. Insoluble materials were removed by centrifugation at 13,000 g at 4uC for 20 min. Affinity chromatography was used as the first step of the purification. Supernatant of M1 and M1N were flowed through a chelating Sepharose Fast Flow Column, and the bonded proteins were eluted with elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole pH 7.9). Supernatant of M1C was applied to a column contained affinity resin (Glutathione Sepharose 4B) and eluted with elution buffer (50 mM Tris, 50 mM reduced glutathione, pH 8.0). It was then changed to Cleavage Buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0), and PreScission Protease and GST-tag were eliminated by passing through the affinity column twice at 4uC followed by further purification by gel filtration using A Superdex 200TM 10/ 30 column (10 mm6300 mm) on an A¨KTA fast protein liquid chromatography system (FPLC). Analytical gel filtration analysis Affinity purified proteins were fractionated by analytical gel filtration on a SuperdexTM 200 HR 10/30 column (10 mm6300 mm). The column was equilibrated with two column of elution buffer before experiment. The process was executed at a flow rate of 0.5 ml/min at constant room temperature of 25uC. Absorbance was monitored at 280 nm, and elution volumes were determined from UV chromatogram. The column was calibrated with the following standard globular proteins: Ribonuclease A (13.7 kDa, 18.8 ml), Cyclophilin A (18.0 kDa, 18.0 ml), Rabbit Action Ovabumin (43 kDa, 16.2 ml), bovine albumin V (68.0 kDa, 15.1 ml) and Aldolase (158 kDa, 13.3 ml). The void volume of column was determined by Blue dextran 2000. The partition coefficient, Kav, was calculated from the elution volume of the sample. A standard calibration curve was obtained by plotting the ratio (Ve2V0)/(Vt2V0) against the logarithm of molecular mass (Ve is elution volume, V0 is void volume and Vt is total bed volume) by UNICORNTM software. The apparent molecular mass of the sample can be estimated based on the acquired straight line. The determination of proteins oligomerization was performed by injection of purified proteins from affinity chromatography onto the gel filtration column at pH 7.4 and pH 5.0. The largest elution volume fraction of M1 or M1N or M1C on gel filtration column was collected and concentrated to a series of concentra- tions in 20 mM Tris, 150 mM NaCl, pH 7.4 or 20 mM sodium acetate, 150 mM NaCl, pH 5.0 by Ultrafiltration. Then 500 ml each sample was load onto the column and eluted with the same buffer to protein solutions. Analytical ultracentrifugation Sedimentation velocity was performed in a Beckman-Coulter XL-I analytical ultracentrifuge. Sample (400 ml) and buffer (400 ml) solutions were loaded into the double sector centerpiece separately, and equipped with a four-cell An-60 Tirotor. 1.2 mg/ ml M1N was equilibrated in 20 mM Tris, 150 mM NaCl pH 7.4 and 1.2 mg/ml M1N in 20 mM sodium acetate, 150 mM NaCl, pH 5.0. 2.8 mg/ml M1C was equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.4. Experiments were carried out with the Influenza A Virus M1 Oligomerization rotor speed of 50,000 rpm at 20uC. Scans were monitored at 280 nm with a radial increment step size of 0.003 cm. Differential sedimentation coefficient distributions, c(s), were calculated by least squares boundary modeling of sedimentation velocity data using the c(s) method, which as an implement of SEDFIT program on www.analyticalultracentrifugation.com/download.htm [58]. The values of apparent sedimentation coefficients (s-values) were corrected to standard conditions (water, 20uC, and infinite dilution) using SEDNTERP program (from www.jphilo.mailway. com/download.htm.) [59], to get the corresponding s-standard value (s20, w). The frictional coefficients (f/f0) and axial ratios (a/b) were calculated by using the vbar method in SEDNTERP program, assuming a prolate ellipsoid model. Sedimentation coefficient distributions was transformed to a molar mass distributions based on the frictional ratio [60]. GST pull-down assays GST pull-down assay was carried out to detect the interactions in M1 molecules. Expression and purification of hisM1, M1N, and M1C used in the experiment were followed with the procedure described above, and purified proteins were exchanged into binding buffer (20 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1% NP40, pH 7.4). The GST-M1 or the control GST protein was expressed in E. coli BL21. Cells were collected and followed by sonication. The cell lysates were centrifuged at 13,000 g for 15 min at 4uC. The supernatants were applied to a column containing Glutathione Sepharose 4B binding for 1 h at 4uC [61]. The column was washed with 20 column volumes of PBS. An equal amount of either GST-M1 or GST bound to affinity resin was mixed with hisM1 or M1N or M1C in 500 ml binding buffer, and then incubated for 2 h at 4uC. The resin was washed six times with washing buffer (300 mM NaCl added in binding buffer,). The quality of proteins used in the assays as following: 10 mg GST-M1 mixed with 20 mg M1N or M1C or hisM1, 10 mg GST mixed with 20 mg M1N or M1C as negative control. Pull down assay was also performed to analyze the effect of pH on the binding activity. 10 mg GST-M1 or GST bound to resin was mixed with 20 mg hisM1 or 20 mg M1N in binding buffer and was washed four times with washing buffer. Then the resin was washed six times in acidic environment (20 mM NaH2PO4, 450 mM NaCl, pH 5.0). Pro- teins bound to the resin were recovered by boiling for 10 min in presence of 26 SDS loading buffer, and subjected to autoradiog- raphy. Proteins were then detected by Western blot with an anti- His-tag monoclonal antibody for hisM1, M1N and an anti-M1 monoclonal antibody for M1C. Glutaraldehyde Cross-linking Protein cross-linking was performed at room temperature. The samples were cross-linked with 0.2% (v/v) glutaraldehyde in the reaction buffer (20 mM sodium phosphate, 100 mM NaCl, pH 8.0). The reaction mixture was incubated for 5 minutes, and then the reaction was quenched by adding Tris (1 M, pH 7.5) to a final concentration of 50 mM. Proteins were analyzed by Tricine- SDS-PAGE CD spectroscopy CD spectra at far-UV (190–260 nm) were performed on a Jasco810 spectropolarimeter at room temperature (25uC). A 1 mm quartz cell was used for far-UV CD spectra. M1 and M1N, which were concentrated to 30 mM by Ultra filtration, were recorded in 50 mM sodium phosphate, pH 7.4. Spectra of 5 mM, 30 mM, 60 mM and 100 mM M1C were measured in 50 mM sodium phosphate, pH 7.4 and 50 mM NaH2PO4, pH 5.0. The spectra were collected at a scanning rate of 1 nm/s with intervals of PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37786