Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Abstract

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

“…Because troponin (48,57), caldesmon (58,59), and myosin (60) all greatly increase the affinity of tropomyosin for actin, we measured the effect of Tmod1 on stTM binding to actin (Fig. 7).…”

The molecular anatomy of caldesmon

“…Measurements of smooth muscle tropomyosin binding to individual caldesmon molecules have been made using ligandsensitive fluorescent labels attached to cysteine on caldesmon or tropomyosin (Cys-190) [95,107,108]. In low-ionic-strength buffers the binding constant is around 106 M-1 but affinity diminishes rapidly with increasing [KCl], reaching 105 M-1 at physiological salt concentrations, indicating largely ionic bonding.…”

“…7). The sequence includes Cys-190 to which the ligand-sensitive fluorescent labels were attached for caldesmon binding measurements [95,107] and 121-173 which was predicted to bind to troponin T [71]. Tropomyosin is, of course, a coiled-coil molecule; the two a-helices are in parallel register so that one binding site would be contributed by two tropomyosin peptides [111,112].…”

“…6; [5,89]), actin increases tropomyosin affinity for caldesmon 5-fold [107] and caldesmon increases tropomyosin affinity for actin (this has been demonstrated with CD! and nonmuscle tropomyosins which have a low affinity for actin [12]).…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…The affinity between CaD and Tm at physiological ionic strength was estimated to be 2.5 × 10 −5 M −1 and was enhanced by actin. 73 Based on further binding studies a model was proposed in which CaD binds Tm in an antiparallel manner at sites near Cys-190 (residue . 74 This region probably is fairly sticky, because it also interacts with calponin.…”