The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% α-helix. The uniformly 15N-labeled M2 protein and both 15N-Val- and 15N-Leu-labeled M2 protein have been expressed from defined M9 media. The 1H-15N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d25 micelles, and the data indicate a homogeneous preparation. The reconstituted M2/ DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for 15N-1H dipolar/15N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25°, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38°). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure.
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