Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV

This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2-4-) in which the Ca2+-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca2+ binding at both Ca2+-binding sites of barnacle TnC did not prevent the incorporation of BTnC2-4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg2+ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca2+-binding sites. Competition experiments between BTnC2-4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC2 indicate that BTnC2-4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC2. These results indicate that disabling sites II and IV changes the affinity of BTnC2-4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament.