PH-Dependent Conformational Changes in the Influenza a M2 Full-Length Protein in Lipid Bilayers

Abstract

Influenza A M2 protein is a homo-tetrameric, membrane protein that conducts protons as one of its multiple functions. Monomeric M2 has an N-terminus that appears to have some b-strand and cysteine disulfide linkages, single transmembrane helix followed by an amphipathic helix and C-terminus whose structure is largely unknown. Together, the TM and amphipathic helices are called ‘the conductance domain”. Even though the conductance domain structure is solved in lipid bilayers, the structure of the full-length protein is not yet fully characterized although considerable spectroscopy of the protein has been published showing that the transmembrane helix within the full length protein is similar to that in the conductance domain.Although, the effect of pH on different truncated versions of the M2 protein has been observed, its effect on the full-length M2 in lipid bilayers is not yet reported. Here, we present how pH affects the opening of the pore in the full-length protein in lipid bilayers and on amantadine block of the wild type M2 channel at pH values that correspond to open and closed states of the channel. These changes in the backbone conformation were determined by unambiguous, inter-monomer distance measurements using different amino acid specific labeled monomers and solid state NMR spectroscopy. In addition, data obtained from the drug resistant mutant (S31N full length) is presented.Distances between the monomers to date have been sparse - these unambiguous restraints not only enhance the structure of the protein surrounding the aqueous pore through the membrane, but in particular there have been no high resolution distance restraints associated with the amphipathic helix until now. Here, we validate the orientation of the amphipathic helix using unambiguous, inter-monomer distance measurements. Effects of environment/conditions (cholesterol/pH) on these measurements will also presented.