caa a22 4500 475744810 CHVBK 20180406123510.0 cr unu---uuuuu 170329e20000801xx s 000 0 eng 10.1023/A:1026589704750 doi (NATIONALLICENCE)springer-10.1023/A:1026589704750 A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin [Elektronische Daten] [Giancarlo Notarianni, Nikolai Gusev, Daniel Lafitte, Tessa Hill, Helen Cooper, Peter Derrick, Steven Marston] Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25°C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state. Kluwer Academic Publishers, 2000 Notarianni Giancarlo Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, SW3 6LY, London, UK aut Gusev Nikolai Department of Biochemistry, School of Biology, Moscow State University, 119899, Moscow, Russia aut Lafitte Daniel Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut Hill Tessa Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut Cooper Helen Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut Derrick Peter Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut Marston Steven Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, SW3 6LY, London, UK aut Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/6(2000-08-01), 537-549 0142-4319 21:6<537 2000 21 10974 https://doi.org/10.1023/A:1026589704750 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026589704750 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Notarianni Giancarlo Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, SW3 6LY, London, UK aut NATIONALLICENCE 700 1- Gusev Nikolai Department of Biochemistry, School of Biology, Moscow State University, 119899, Moscow, Russia aut NATIONALLICENCE 700 1- Lafitte Daniel Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut NATIONALLICENCE 700 1- Hill Tessa Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut NATIONALLICENCE 700 1- Cooper Helen Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut NATIONALLICENCE 700 1- Derrick Peter Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, CV4 7AL, Coventry, UK aut NATIONALLICENCE 700 1- Marston Steven Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, SW3 6LY, London, UK aut NATIONALLICENCE 773 0- Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/6(2000-08-01), 537-549 0142-4319 21:6<537 2000 21 10974 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer