title: Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C.
abstract: 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 indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.
authors.affiliation: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore 21201, USA.
authors.forename: J
authors.initials: J
authors.lastname: Digel
authors.forename: O
authors.initials: O
authors.lastname: Abugo
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Z
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
authors.forename: J H
authors.initials: JH
authors.lastname: Collins
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D007454:Iodides / Q000737:chemistry; D008274:Magnesium / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D007454:Iodides; D019209:Troponin C; D019210:Troponin I; D020106:Acrylamide; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium