Influenza A Virus M1 Oligomerization Figure 4. Sedimentation velocity analytical ultracentrifugation determined the oligomerization statue of the N-terminal domain. (A and C) The panels showed the sedimentation coefficient distributions c(s) of purified M1N from the 17.6-ml fraction at pH 7.4 (A), and from the 17.4- ml fraction at pH 5.0 (C). The data were acquired at a protein concentration of 1.2 mg/ml. (B and D) The molecular mass distribution derived from c(s) data of panel A and C, respectively. doi:10.1371/journal.pone.0037786.g004 range of protein concentrations at both neutral (Fig. 7A) and acidic pH (data not shown). In all case, nearly identical curves were obtained, indicating the consistency of the secondary structure in this domain. The spectrum of M1C showed a clear minimum at 203 nm and a weak minimum at 222 nm. The minimum at 222 nm was indicative of a-helix content, while the minimum at 203 nm could be the result of a combination of a-helix (208 nm) and random coil structure (200 nm). The exact secondary structure composition of the three proteins was further analyzed by DICHROWEB, with the above data converted into mean molar ellipticity per residue. The results showed that the C- terminal domain is composed of 31% a-helix and 69% random coil. Thermal-induced denaturation of M1C measured by recording ellipticity at 222 nm further revealed the possible heat-induced conformation change. The data showed a three- state transition with the apparent presence of intermediate(s) during denaturation. The two transition points were 32uC and 69uC (Fig. 7B). The content of a-helix was greatly decreased while that of certain random coil structure was induced with the increase of temperature. To validate this, M1C was incubated at two temperature points, and then analyzed by CD (Fig. 7C). At 60uC, the ellipticity value at 222 nm increased, and the other minimum moved from 203 nm to 201 nm, both representing the loss of helix structure. Moreover, the denaturation of M1C was found to be fully reversible, as almost identical CD spectra were obtained for native refolded M1C that were cooled from 90uC to 25uC. To characterize the hydrophobic cluster of M1C, the transfor- mation of 4,4-Dianilino-1,1-binaphthyl-5,5-disulphonic acid (ANS) fluorescence spectra was investigated. ANS is a fluorescent probe that has been widely used to monitor the hydrophobic exposure of proteins. Fluorescence of ANS is weak in polar solvents, but is enhanced through the association with exposed hydrophobic region of proteins. As shown in Fig. 7D, compared to ANS probe alone, no obvious ANS fluorescence intensity change was observed when adding dimeric M1C, which suggested that M1C has a hydrophobic core and that little hydrophobic area was exposed at 25uC. In contrast, the fluorescence intensity of ANS was slightly enhanced when M1C was heated at 37uC, and its emission maximum was blue-shifted. Therefore, this result also indicated that the hydrophobic core of M1C is exposed to solution at 37uC, which is higher than the first transition point. PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37786