melitten and 2-(4-toluidino)-6-naphthalenesulfonic-acid

Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.

Topics: Binding Sites; Calcium; Calcium-Binding Proteins; Calmodulin; Cations, Divalent; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Humans; Magnesium; Melitten; Naphthalenesulfonates; Peptide Fragments; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Skin; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Transglutaminases; Tryptophan; Tyrosine

Centrin and calmodulin (CaM) are closely related four-EF-hand Ca(2+)-binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca(2+) per dimer, likely to site IV in each monomer. Ca(2+) binding to centrin 2 displays pronounced negative cooperativity and a [Ca(2+)](0.5) of 30 microM. As in CaM, Ca(2+) binding leads to the exposure of a hydrophobic probe-accessible patch on the surface of centrin 2. Provided Ca(2+) is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca(2+). Our data point to surprising differences in the mode of activation of these homologous proteins.

Topics: Calcium; Calcium-Binding Proteins; Cations, Divalent; Chromosomal Proteins, Non-Histone; Fluorescent Dyes; Humans; Magnesium; Melitten; Naphthalenesulfonates; Peptides; Protein Structure, Secondary; Protein Structure, Tertiary

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.

Topics: Bee Venoms; Fluorescent Dyes; Kinetics; Melitten; Naphthalenesulfonates; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet