Characterization of a calcium‐dependent calmodulin‐binding domain in the 135‐kD human protein 4.1 isoform

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Abstract
The putative calmodulin binding domain of non-erythroid protein 4.1, previously suggested by Kelly et al. [Kelly, G. M., Zelus, B. D. & Moon, R. T. (1991) J. Biol. Chem. 266, 12469-12473] has been synthesized, and its binding to calmodulin has been studied by fluorescence spectroscopy. For this purpose, the peptide has been N-terminally dansylated. The 4.1 peptide Dns-Abu-S76RGLSRLFSSFLKRPKS92, binds calmodulin in a calcium-dependent way with high affinity (Kd = 23 +/- 6 nM). The peptide inhibits bovine-heart phosphodiesterase with an IC50 of 50 nM. Since the sequence of the peptide shows two putative consensus sites of phosphorylation by cAMP-dependent protein kinase or Ca2+-calmodulin protein-kinase II, the interaction of the two mono-phosphorylated peptides (P4.1 Ser(80-P) and P4.1 Ser(84-P)) and the di-phosphorylated peptide (P4.1 Ser(80-P)/Ser(84-P)) with calmodulin has been investigated. A decrease of affinity by a factor 1.5-8 has been observed for the phosphorylated peptides. CD measurements have shown an increase of the content of alpha helices in the peptides when bound to calmodulin.

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