title: NMR determination of protein partitioning into membrane domains with different curvatures and application to the influenza M2 peptide.
abstract: The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use (31)P and (13)C solid-state NMR to examine M2-induced membrane curvature. M2(22-46), which includes only the transmembrane (TM) helix, and M2(21-61), which contains an additional amphipathic helix, are studied. (31)P chemical shift lineshapes indicate that M2(21-61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22-46) has minimal effect. The lamellar and isotropic domains have distinct (31)P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. (31)P- and (13)C-detected (1)H T(2) relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21-61) preferentially binds to the high-curvature domain. (31)P linewidths indicate that the isotropic vesicles induced by M2(21-61) are 10-35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides.
authors.affiliation: Department of Chemistry, Iowa State University, Ames, Iowa, USA.
authors.forename: Tuo
authors.initials: T
authors.lastname: Wang
authors.forename: Sarah D
authors.initials: SD
authors.lastname: Cady
authors.forename: Mei
authors.initials: M
authors.lastname: Hong
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D002784:Cholesterol; D004134:Dimyristoylphosphatidylcholine; D008051:Lipid Bilayers; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins