The interaction of calmodulin with amphiphilic peptides

J Biol Chem. 1985 Feb 25;260(4):2527-34.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids
  • Animals
  • Bacterial Proteins / metabolism
  • Binding, Competitive
  • Brain / enzymology
  • Calmodulin / metabolism*
  • Cattle
  • Circular Dichroism
  • Fluorenes*
  • Hemolysin Proteins
  • Melitten / metabolism
  • Oligopeptides / metabolism
  • Peptides / metabolism*
  • Phosphoric Diester Hydrolases / metabolism
  • Sepharose

Substances

  • Amino Acids
  • Bacterial Proteins
  • Calmodulin
  • Fluorenes
  • Hemolysin Proteins
  • N(alpha)-fluorenylmethyloxycarbonylamino acids
  • Oligopeptides
  • Peptides
  • Melitten
  • delta hemolysin protein, Staphylococcus aureus
  • Sepharose
  • Phosphoric Diester Hydrolases