Calcium- and Magnesium-Dependent Interactions between the C-Terminus of Troponin I and the N-Terminal, Regulatory Domain of Troponin C

• Published in: Archives of biochemistry and biophysics 2001-03, Vol.387 (2), p.243-249
• Main Authors: Digel, Jeanne, Abugo, Omoefe, Kobayashi, Tomoyoshi, Gryczynski, Zygmunt, Lakowicz, Joseph R., Collins, John H.
• Format: Article
• Language: English
• Subjects: Acrylamide - chemistry; Amino Acid Substitution; Animals; Binding Sites - physiology; calcium; Calcium - metabolism; fluorescence; Iodides - chemistry; Magnesium - metabolism; muscle; Muscle Contraction - physiology; Mutagenesis, Site-Directed; Protein Structure, Tertiary - physiology; Rabbits; regulation; Sequence Deletion; Spectrometry, Fluorescence; troponin; Troponin C - chemistry; Troponin C - genetics; Troponin C - metabolism; Troponin I - metabolism; Tryptophan - chemistry; Tryptophan - genetics
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2000.2259

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central 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 this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with 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 binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites 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 differences between full-length TnI and TnI(1–159) in their effect on Trp-26. Our results provide the first indication that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regulatory domain of TnC.