Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1
2001
Vetter, Stefan W. | Leclerc, Estelle
We have previously characterized the calciumâdependent calmodulin (CaM)âbinding domain (Ser76–Ser92) of the 135âkDa 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 17âresidue peptide alters their ability to adopt α helical conformation in a positionâdependent manner. The helical content of the peptides was determined by CDâspectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the diâphosphorylated 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 α helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaMâbinding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptideâbinding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.
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