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The binding of 14‐3‐3γ to membranes studied by intrinsic fluorescence spectroscopy
Author(s) -
Bustad Helene J.,
Underhaug Jarl,
Halskau Øyvind,
Martinez Aurora
Publication year - 2011
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2011.03.027
Subject(s) - fluorescence , membrane , chemistry , fluorescence spectroscopy , tryptophan , biophysics , fluorescence correlation spectroscopy , bilayer , fluorescence anisotropy , membrane protein , binding site , biochemistry , biology , amino acid , molecule , organic chemistry , physics , quantum mechanics
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.