Expression and Initial Structural Insights from Solid-State NMR of the M2 Proton Channel from Influenza A Virus

• Published in: Biochemistry (Easton) Vol. 41; no. 37; pp. 11294 - 11300
• Main Authors: Tian, Changlin, Tobler, Kurt, Lamb, Robert A, Pinto, Lawrence H, Cross, T. A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17-09-2002
• Subjects: Dimyristoylphosphatidylcholine - chemistry; Influenza A virus; Influenza A virus - chemistry; Ion Channels - biosynthesis; Ion Channels - chemistry; Ion Channels - genetics; Liposomes; Nuclear Magnetic Resonance, Biomolecular - methods; Phosphatidylglycerols - chemistry; Phosphorus Isotopes; Protein Structure, Secondary; Protons; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Solubility; Viral Matrix Proteins - biosynthesis; Viral Matrix Proteins - chemistry; Viral Matrix Proteins - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi025695q

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-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 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.