Characterization of a calcium-dependent calmodulin-binding domain in the 135-kD human protein 4.1 isoform.
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.
Leclerc
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Institute of Molecular Biology and Biophysics, ETH-Hönggerberg, Zürich, Switzerland.
Vetter
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D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D003598:Cytoskeletal Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009479:Neuropeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D013050:Spectrometry, Fluorescence
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D010726:Phosphodiesterase Inhibitors; D010748:Phosphopeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D010768:Phosphoserine; D000242:Cyclic AMP; D002118:Calcium