Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1

Eur J Biochem. 2001 Aug;268(15):4292-9. doi: 10.1046/j.1432-1327.2001.02347.x.


We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arginine / chemistry
  • Binding Sites
  • Calmodulin / chemistry*
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Cytoskeletal Proteins*
  • Humans
  • Membrane Proteins / chemistry*
  • Models, Molecular
  • Models, Theoretical
  • Neuropeptides*
  • Peptide Biosynthesis
  • Peptides / chemistry
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Protein Isoforms
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Serine / chemistry*
  • Spectrometry, Fluorescence


  • Calmodulin
  • Cytoskeletal Proteins
  • Membrane Proteins
  • Neuropeptides
  • Peptides
  • Protein Isoforms
  • erythrocyte membrane band 4.1 protein
  • erythrocyte membrane protein band 4.1-like 1
  • Serine
  • Arginine