title: Dual-fluorescence L-amino acid reports insertion and orientation of melittin peptide in cell membranes.
abstract: Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an l-amino acid (AFaa) containing a dual-fluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane. This concept was validated by replacing the leucine-9 (L9) and tryptophan-19 (W19) residues by AFaa in melittin, a well-studied membrane-active peptide. Due to high sensitivity of AFaa dual emission to the environment polarity, we detected a much deeper insertion of L9 peptide position into the bilayer, compared to the W19 position. Moreover, using fluorescence microscopy with a polarized light excitation, we found different orientation of AFaa at L9 and W19 positions of melittin in the bilayers of giant vesicles and cellular membranes. These results suggested that in the natural membranes, similarly to the model lipid bilayers, melittin is preferentially oriented parallel to the membrane surface. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie , 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors.forename: Viktoriia Y
authors.initials: VY
authors.lastname: Postupalenko
authors.forename: Oleksandr M
authors.initials: OM
authors.lastname: Zamotaiev
authors.forename: Volodymyr V
authors.initials: VV
authors.lastname: Shvadchak
authors.forename: Aleksandr V
authors.initials: AV
authors.lastname: Strizhak
authors.forename: Vasyl G
authors.initials: VG
authors.lastname: Pivovarenko
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Klymchenko
authors.forename: Yves
authors.initials: Y
authors.lastname: Mely
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D015195:Drug Design; D005419:Flavonoids / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008555:Melitten / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D017433:Protein Structure, Secondary
chemical_list: D000596:Amino Acids; D005419:Flavonoids; D005456:Fluorescent Dyes; D008555:Melitten; C041477:3-hydroxyflavone