A calmodulin-binding peptide of caldesmon.
Caldesmon is a major actin-binding protein identified in smooth muscle and many non-muscle cells. It also interacts with calmodulin and a number of other acidic proteins. We have shown previously that the polypeptide stretch from Val629 to Ser666 near the C terminus contains a calmodulin binding site (Wang, C.-L. A., Wang, L.-W. C., Xu, S., Lu, R. C., Saavedra-Alanis, V., and Bryan, J. (1991) J. Biol. Chem. 266, 9166-9172). On the other hand, Bartegi et al. (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) reported a cyanogen bromide fragment beginning at Trp659 which is also capable of binding both calmodulin and actin. A comparison of the overlapping sequence between these two peptides suggests that this calmodulin binding site is localized in a 7-residue segment, 659Trp-Glu-Lys-Gly-Asn-Val-Phe665. We have chemically synthesized an 18-residue peptide (GS17C, from Gly651 to Ser667 with an added cysteine at the C terminus) that contains this segment. This peptide was purified by high performance liquid chromatography and labeled with fluorescent probes at the terminal cysteine residue. We found that GS17C indeed binds calmodulin in a Ca(2+)-dependent manner (Kd = 8 x 10(-7) M) and appears to compete with caldesmon. Interestingly, this synthetic peptide also co-sediments with F-actin, binding to actin being displaceable by calmodulin, as in the case of the native caldesmon. But GS17C does not have any effect on the actomyosin ATPase activity, indicating that this peptide segment does not contain the inhibitory region.
Zhan
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Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Wong
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Wang
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D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D002846:Chromatography, Affinity; D005895:Gizzard, Avian / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D010455:Peptides