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

We have previously characterized the calcium-dependent calmodulin (CaM)-bin ding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fl uorescence spectroscopy and chemically synthesized nonphosphorylated or ser ine phosphorylated peptides [Leclerc, E. & Vetter, S. ( 1998) Eur J. Bioche m. 258, 567-671]. Here we demonstrate that phosphorylation of two serine re sidues within the 17-residue peptide alters their ability to adopt alpha he lical conformation in a position-dependent manner. The helical content of t he 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 Ser8 4 phosphorylated peptides the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that in itial helix formation involves the central residues Leu82-Phe86. The abilit y of the peptides to adopt alpha helical conformations did not correlate wi th 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 in troduced into the CaM peptide-binding interface by the phosphate groups, ra ther than being due to the effect of phosphorylation on the secondary struc ture of the peptides.