CONCERTED ACTION OF THE HIGH-AFFINITY CALCIUM-BINDING SITES IN SKELETAL-MUSCLE TROPONIN-C

Mutants of each of the four divalent cation binding sites of chicken s keletal muscle troponin C (TnC) were constructed using site directed m utagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site inte ractions both within and between the N- and C-terminal domains, in thi s study the mutants are examined for their ability to associate with o ther components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in re constitution assays are largely confined to the domain in which it occ urs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase acti vity, whereas the mutants of sites III and IV regulate activity but ar e unable to remain bound to thin filaments unless Ca2+ is present. Whe n all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, al lowing it to attach firmly to thin filaments. Calcium can replace Mg2 at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC c omplex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tensi on threshold) are required to effect this transformation than in the r ecombinant wild-type protein, suggesting that the mutants reveal an at tachment mediated by Ca2+ in the N-domain sites.