Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly

Biochemistry. 1999 Sep 14;38(37):11905-13. doi: 10.1021/bi990720m.


The M2 protein from influenza A virus is a 97-residue homotetrameric membrane protein that functions as a proton channel. To determine the features required for the assembly of this protein into its native tetrameric state, the protein was prepared by total synthesis using native chemical ligation of unprotected peptide segments. Circular dichroism spectroscopy of synthetic M2 protein in dodecylphosphocholine (DPC) micelles indicated that approximately 40 residues were in an alpha-helical secondary structure. The tetramerization of the full-length protein was compared to that of a 25-residue transmembrane (TM) fragment. Analytical ultracentrifugation demonstrated that both the peptide and the full-length protein in DPC micelles existed in a monomer-tetramer equilibrium. Comparison of the association constants for the two sequences showed the free energy of tetramerization of the full-length protein was more favorable by approximately 7 kcal/mol. Partial proteolysis of DPC-solubilized M2 was used as a further probe of the structure of the full-length protein. A 15-20-residue segment C-terminal to the membrane-spanning region was found to be highly resistant to digestion by chymotrypsin and trypsin. This region, which we have modeled as an extension of the TM helices, may help to stabilize the tetrameric assembly.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chymotrypsin / metabolism
  • Circular Dichroism
  • Hydrolysis
  • Influenza A virus / chemistry*
  • Ligands
  • Membrane Proteins / chemical synthesis*
  • Membrane Proteins / metabolism
  • Membrane Proteins / physiology*
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemical synthesis*
  • Peptide Fragments / metabolism
  • Peptide Fragments / physiology*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / metabolism
  • Ultracentrifugation
  • Viral Matrix Proteins / chemical synthesis*
  • Viral Matrix Proteins / metabolism
  • Viral Matrix Proteins / physiology*


  • Ligands
  • M-protein, influenza virus
  • M2 protein, Influenza A virus
  • Membrane Proteins
  • Peptide Fragments
  • Viral Matrix Proteins
  • Chymotrypsin
  • Trypsin