Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin–myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175 → alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin–tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure–function relationships of calponin and its regulation by phosphorylation.
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Research Article|
August 23 2000
A role for serine-175 in modulating the molecular conformation of calponin
Jian-Ping JIN;
Jian-Ping JIN
1
*Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970, U.S.A.
1To whom correspondence should be addressed (e-mail jxj12@po.cwru.edu).
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Michael P. WALSH;
Michael P. WALSH
†Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada
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Cindy SUTHERLAND;
Cindy SUTHERLAND
†Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada
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Wenhua CHEN
Wenhua CHEN
*Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970, U.S.A.
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Publisher: Portland Press Ltd
Received:
January 04 2000
Revision Received:
May 04 2000
Accepted:
May 22 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 579–588.
Article history
Received:
January 04 2000
Revision Received:
May 04 2000
Accepted:
May 22 2000
Citation
Jian-Ping JIN, Michael P. WALSH, Cindy SUTHERLAND, Wenhua CHEN; A role for serine-175 in modulating the molecular conformation of calponin. Biochem J 1 September 2000; 350 (2): 579–588. doi: https://doi.org/10.1042/bj3500579
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