Conformational studies of aqueous melittin. Characteristics of a fluorescent probe binding site.
The structural basis of the interaction of melittin with amphipathic molecular assemblies, i.e. membranes, was investigated by studying the binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to melittin by ultraviolet and fluorescence spectroscopy. Monomeric melittin did not significantly bind TNS as judged by UV and fluorescent spectroscopy. Tetrameric melittin bound two TNS molecules per protomer with dissociation constants (Kd) of 4.2 X 10(-6) M. TNS binding to tetrameric melittin led to an increase in fluorescence quantum yield of 180-fold over the value for TNS alone in aqueous buffer (phi H2O = 0.004). Five independent experimental findings suggest that the arginine residues of melittin provide one portion of the TNS binding site (presumably by formation of an especially stable "ringed-structure" salt bridge between the tetrahedral sulfonyl anion of TNS and the argininyl residues of melittin): 1) the Kd for binding is independent of pH from 6.0 to 10.8, the range in which the alpha-aminoglycine and epsilon-aminolysines titrate; 2) TNS binding fails to perturb the kinetics of the reaction of 2,4,6-trinitrobenzenesulfonate with Lys-21 or Lys-23; 3) 1,7,21,23-acetyl-melittin, in which the NH2 terminus and all lysines are acetylated, binds TNS with a Kd similar to that for normal melittin; 4) guanidination of the NH2-terminal glycines and lysines of melittin (forming N-guanidoglycine and homoargininyl residues, respectively) increases the number of TNS molecules bound per protomer to approximately 5; 5) conversion of Arg-22 and Arg-24 to the anionic N7, N8-(2,3-dihydro(7,7-dimethyl))bicyclo[2.2.1] heptane - 1 - methanesulfonylborate complex abolishes TNS binding, as judged by fluorescence.
D001514:Bee Venoms; D005456:Fluorescent Dyes; D007700:Kinetics; D008555:Melitten; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001514:Bee Venoms; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D008555:Melitten; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid