title: The interaction of calmodulin with amphiphilic peptides.
abstract: Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.
authors.forename: J A
authors.initials: JA
authors.lastname: Cox
authors.forename: M
authors.initials: M
authors.lastname: Comte
authors.forename: J E
authors.initials: JE
authors.lastname: Fitton
authors.forename: W F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D000596:Amino Acids; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D005449:Fluorenes; D006460:Hemolysin Proteins; D008555:Melitten / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012685:Sepharose
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002147:Calmodulin; D005449:Fluorenes; D006460:Hemolysin Proteins; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009842:Oligopeptides; D010455:Peptides; D008555:Melitten; C025994:delta hemolysin protein, Staphylococcus aureus; D012685:Sepharose; D010727:Phosphoric Diester Hydrolases