title: The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy.
abstract: Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway.
authors.forename: Helene J
authors.initials: HJ
authors.lastname: Bustad
authors.forename: Jarl
authors.initials: J
authors.lastname: Underhaug
authors.forename: Oyvind
authors.initials: O
authors.lastname: Halskau
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D014364:Tryptophan