A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibit ory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smoo th muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin a re not Ca2+-regulated at 25 degreesC/50 mM KCl. We isolated the Ca2+-bindin g protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chroma tography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyos in and caldesmon to reconstitute a normally regulated thin filament at 25 d egreesC/50 mM KCl. It reverses caldesmon inhibition at pCa5 under condition s where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon i ndependently of [Ca2+]. Amino acid sequencing, and electrospray mass spectr ometry 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 spectrom etry and nozzle skimmer fragmentation patterns are different indicating a l ess stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evide nce for the presence of any covalent or non-covalently bound adduct. The on ly remaining conclusion is that CaBP is calmodulin locked in a metastable a ltered state.