Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus.
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% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) 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 (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). 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.
Tian
Changlin
C
National High Magnetic Field Lab, Institute of Molecular Biophysics, and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32310, USA.
Tobler
Kurt
K
Lamb
Robert A
RA
Pinto
Lawrence H
LH
Cross
T A
TA
D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D009980:Influenza A virus / Q000737:chemistry*; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008081:Liposomes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010715:Phosphatidylglycerols / Q000737:chemistry; D010759:Phosphorus Isotopes; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000086082:Viroporin Proteins
D004134:Dimyristoylphosphatidylcholine; D007473:Ion Channels; D008081:Liposomes; D010715:Phosphatidylglycerols; D010759:Phosphorus Isotopes; D011522:Protons; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C002773:dimyristoylphosphatidylglycerol