UTILIZATION OF TROPONIN-C AS A MODEL CALCIUM-BINDING PROTEIN FOR MAPPING OF THE CALMODULIN-BINDING SITES OF CALDESMON

Troponin C, a structural analogue of calmodulin, was used for mapping the calmodulin-binding sites of caldesmon. The apparent K-d values for the formation of the caldesmon-calcium-binding-protein complex as det ermined by native gel electrophoresis were 0.5, 1.2 and 3.9 mu M for c almodulin, rabbit skeletal muscle troponin C and bovine cardiac tropon in C respectively. Troponin C induced a 4-6 nm blue shift of the Trp f luorescence of caldesmon without affecting the amplitude of fluorescen ce. In the presence of Ca2+, troponin C induced partial displacement o f caldesmon from actin-tropomyosin complexes. Addition of 5,5'-dithiob is(nitrobenzoic) acid to an equimolar complex of caldesmon and troponi n C induced disulphide cross-linking between Cys-98 of rabbit skeletal muscle troponin C and the single Cys residue of duck gizzard caldesmo n, located in a position analogous to Cys-580 of the chicken gizzard p rotein. The cross-linked caldesmon-troponin C complex was ineffective in inhibiting actomyosin ATPase activity. It is concluded that Cys-580 of caldesmon can be located close to both the central helix of calciu m-binding proteins and the C-terminal domain of actin. This may be imp ortant for the regulation of actomyosin ATPase activity by caldesmon.