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

Jeanne Digel, Omoefe Abugo, Tomoyoshi Kobayashi, Zygmunt Gryczynski, Joseph R. Lakowicz, John H. Collins, Jeanne Digel, Omoefe Abugo, Tomoyoshi Kobayashi, Zygmunt Gryczynski, Joseph R. Lakowicz, John H. Collins

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish
Pages (from-to)243-249
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume387
Issue number2
DOIs
StatePublished - 15 Mar 2001

Fingerprint

Troponin C
Troponin I
Magnesium
Calcium
Troponin
Muscle
Striated Muscle
Binding Sites
Vertebrates
Amino Acids
Divalent Cations
Myosins
Muscle Contraction
Actins
Quenching
Fluorescence

Keywords

  • Calcium
  • Fluorescence
  • Muscle
  • Regulation
  • Troponin

Cite this

Digel, Jeanne ; Abugo, Omoefe ; Kobayashi, Tomoyoshi ; Gryczynski, Zygmunt ; Lakowicz, Joseph R. ; Collins, John H. ; Digel, Jeanne ; Abugo, Omoefe ; Kobayashi, Tomoyoshi ; Gryczynski, Zygmunt ; Lakowicz, Joseph R. ; Collins, John H. / Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C. In: Archives of Biochemistry and Biophysics. 2001 ; Vol. 387, No. 2. pp. 243-249.
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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 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.",
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Digel, J, Abugo, O, Kobayashi, T, Gryczynski, Z, Lakowicz, JR, Collins, JH, Digel, J, Abugo, O, Kobayashi, T, Gryczynski, Z, Lakowicz, JR & Collins, JH 2001, 'Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C', Archives of Biochemistry and Biophysics, vol. 387, no. 2, pp. 243-249. https://doi.org/10.1006/abbi.2000.2259

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C. / Digel, Jeanne; Abugo, Omoefe; Kobayashi, Tomoyoshi; Gryczynski, Zygmunt; Lakowicz, Joseph R.; Collins, John H.; Digel, Jeanne; Abugo, Omoefe; Kobayashi, Tomoyoshi; Gryczynski, Zygmunt; Lakowicz, Joseph R.; Collins, John H.

In: Archives of Biochemistry and Biophysics, Vol. 387, No. 2, 15.03.2001, p. 243-249.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

AU - Digel, Jeanne

AU - Abugo, Omoefe

AU - Kobayashi, Tomoyoshi

AU - Gryczynski, Zygmunt

AU - Lakowicz, Joseph R.

AU - Collins, John H.

AU - Digel, Jeanne

AU - Abugo, Omoefe

AU - Kobayashi, Tomoyoshi

AU - Gryczynski, Zygmunt

AU - Lakowicz, Joseph R.

AU - Collins, John H.

PY - 2001/3/15

Y1 - 2001/3/15

N2 - 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.

AB - 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.

KW - Calcium

KW - Fluorescence

KW - Muscle

KW - Regulation

KW - Troponin

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U2 - 10.1006/abbi.2000.2259

DO - 10.1006/abbi.2000.2259

M3 - Article

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