The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform gamma. 14-3-3 gamma 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 14-3-3 gamma mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which p-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 14-3-3 gamma to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3 gamma, and that Trp residues do not intercalate in the bilayer. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.