Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of ver tebrate striated muscle by conferring Ca2+ sensitivity to the interaction o f actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contai ns two homologous domains and four divalent cation binding sites. Two struc tural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and t wo regulatory sites in the N-terminal domain are specific for Ca2+. Interac tions between TnC and the inhibitory Tn subunit troponin I (TnI) are of cen tral importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In t his report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and qu enching measurements to monitor Ca2+- and Mg2+-dependent changes in the env ironment of Trp-26 in isolated TnC, as well as in binary complexes of TnC w ith a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159) , which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four bind ing sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding si tes in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant diffe rences between full-length TnI and TnI(1-159) in their effect on Trp-26, Ou r results provide the first indication that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through C a2+-dependent interactions with the regulatory domain of TnC. (C) 2001 Acad emic Press.