Regulation of fast-twitch fibers was completely blocked with the CBM2A mutant where both trigger sites were inactive, indicating structural specification of the entire EF-hand motif is needed.
Structural specification of the entire EF-hand motif of site 1, not just Ca2+ coordination, is required to fully operate the Ca2+ switch in fast-twitch fibers.
Structure/function relationships in troponin C are studied with vertebrate fast-twitch fibers by exchanging the skeletal troponin C with two bacterially synthesized recombinant proteins designed by site-directed mutagenesis of cardiac troponin C. One mutant (CBM1) contained an additional active site, by deleting Val-28 and converting Leu-29, Gly-30, Ala-31 and Glu-32 to Asp, Ala, Asp and Gly, respectively, in the normally inactive trigger site 1 in the N-terminus. In another mutant (CBM2A), the normally active site 2 was inactivated by conversion of Asp-65 to Ala. The fibers were found to be down-regulated with recombinant cardiac troponin C (CTnC3), as with tissue-cardiac-troponin-C. With mutants, in one case (CBM1) the regulation was unmodified despite Ca2+ coordination by all sites. In contrast, regulation was found to be completely blocked with the mutant (CBM2A) where both trigger sites were inactive. The results provide the first indication that structural specification of the entire EF-hand motif of site 1, and not just Ca2+ coordination, is needed to operate fully the Ca2+ switch in fast-twitch fibers.
Gulati et al. (1989) studied this question. Recombinant cardiac troponin C mutants (CBM1, CBM2A) vs. Tissue-cardiac-troponin-C was evaluated on Regulation of fast-twitch skeletal muscle fibers. Regulation of fast-twitch fibers was completely blocked with the CBM2A mutant where both trigger sites were inactive, indicating structural specification of the entire EF-hand motif is needed.