Functional Characterization of the Last 5 Residues of the C Terminus in Cardiac Troponin I

Abstract

The C-terminus of cardiac troponin I (cTnI) is a highly conserved region of the protein. Previous reports have suggested that the last 17 residues at the C-terminal end of cTnI do not directly interact with cardiac troponin C (cTnC). However, a 17 residue C-terminal deletion in human cTnI is associated with myocardial stunning, and was previously found to increase calcium sensitivity in an in vitro motility assay (Foster et al., Circ. Res. 2003;93:917-924). To further investigate this region of cTnI, we generated three C-terminal deletion mutations in human cTnI: del1 (deletion of residue 210), del3 (deletion of residues 208-210), and del5 (deletion of residues 206-210). A monocysteine mutant of cTnC (C35S) was purified and labeled with the fluorescent probe 2-[4’-(iodoacetamido) anilino] naphthalene-6-sulfonic acid (IAANS) at Cys-84. Upon reconstitution of the labeled cTnC with cardiac troponin T (cTnT) and truncated or wild-type cTnI to form troponin complexes, the calcium-dependent changes in fluorescence were measured. The results show that the troponin complex with the cTnI del5 mutation had increased calcium affinity (P<0.05); while the cTnI del1- and del3 troponin complexes showed no significant difference in calcium affinity when compared to wild-type troponin. Mammalian two-hybrid studies showed that the interaction between cTnC and cTnI deletion mutants were impaired in del3 and del5 mutants when compared to wild-type cTnI. Two-hybrid studies also showed that the interaction between cTnT and the cTnI del5 mutant was impaired when compared to wild-type cTnI or the other deletion mutants. Our results suggest that the last 5 C-terminal residues of cTnI are important for the physiological functions of cTnI and directly influences the binding of cTnI with cTnC and cTnT.