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Down‐regulation of fast‐twitch skeletal muscle fiber with cardiac troponin‐C and recombinant mutants Structure/function studies with site‐directed mutagenesis
Author(s) -
Gulati Jagdish,
Babu Árvind,
Putkey John A.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80420-x
Subject(s) - troponin c , mutant , troponin complex , mutagenesis , recombinant dna , site directed mutagenesis , chemistry , troponin t , troponin i , troponin , biochemistry , skeletal muscle , microbiology and biotechnology , biology , gene , anatomy , medicine , myocardial infarction , psychiatry
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 Ca 2+ 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 Ca 2+ coordination, is needed to operate fully the Ca 2+ switch in fast‐twitch fibers.