Inhibitory region of troponin I: Ca2+-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

Tomoyoshi Kobayashi, Minae Kobayashi, Zygmunt Gryczynski, Joseph R. Lakowicz, John H. Collins

Research output: Contribution to journalArticle

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Abstract

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys- 117 and labeled them with the thiol-specific fluorescent probe N- (iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca2+ by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin- tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca2+, reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 Å for Cys-96 and 35.2 Å for Cys-117. In the presence of Ca2+, Cys-96 moved away from actin Cys-374 by ~3.6 Å, while Cys-117 moved away by ~8 Å. This suggests the existence of a flexible 'hinge' region near the middle of TnI, allowing amino acid residues in the N- terminal half of TnI to interact with TnC in a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.

Original languageEnglish
Pages (from-to)86-91
Number of pages6
JournalBiochemistry
Volume39
Issue number1
DOIs
StatePublished - 11 Jan 2000

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Troponin I
Troponin C
Actins
ethylenediamine
Labels
Fluorescence Resonance Energy Transfer
Acrylamide
Anisotropy
Hinges
troponin-tropomyosin complex
Fluorescent Dyes
Sulfhydryl Compounds
Muscle
Quenching
Fluorescence
Amino Acids
Muscles

Cite this

@article{d91d91bafb444d7eaabae59bc1584eab,
title = "Inhibitory region of troponin I: Ca2+-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments",
abstract = "In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys- 117 and labeled them with the thiol-specific fluorescent probe N- (iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca2+ by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin- tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca2+, reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 {\AA} for Cys-96 and 35.2 {\AA} for Cys-117. In the presence of Ca2+, Cys-96 moved away from actin Cys-374 by ~3.6 {\AA}, while Cys-117 moved away by ~8 {\AA}. This suggests the existence of a flexible 'hinge' region near the middle of TnI, allowing amino acid residues in the N- terminal half of TnI to interact with TnC in a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.",
author = "Tomoyoshi Kobayashi and Minae Kobayashi and Zygmunt Gryczynski and Lakowicz, {Joseph R.} and Collins, {John H.}",
year = "2000",
month = "1",
day = "11",
doi = "10.1021/bi991903b",
language = "English",
volume = "39",
pages = "86--91",
journal = "Biochemistry",
issn = "0006-2960",
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}

Inhibitory region of troponin I : Ca2+-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments. / Kobayashi, Tomoyoshi; Kobayashi, Minae; Gryczynski, Zygmunt; Lakowicz, Joseph R.; Collins, John H.

In: Biochemistry, Vol. 39, No. 1, 11.01.2000, p. 86-91.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Inhibitory region of troponin I

T2 - Ca2+-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

AU - Kobayashi, Tomoyoshi

AU - Kobayashi, Minae

AU - Gryczynski, Zygmunt

AU - Lakowicz, Joseph R.

AU - Collins, John H.

PY - 2000/1/11

Y1 - 2000/1/11

N2 - In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys- 117 and labeled them with the thiol-specific fluorescent probe N- (iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca2+ by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin- tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca2+, reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 Å for Cys-96 and 35.2 Å for Cys-117. In the presence of Ca2+, Cys-96 moved away from actin Cys-374 by ~3.6 Å, while Cys-117 moved away by ~8 Å. This suggests the existence of a flexible 'hinge' region near the middle of TnI, allowing amino acid residues in the N- terminal half of TnI to interact with TnC in a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.

AB - In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys- 117 and labeled them with the thiol-specific fluorescent probe N- (iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca2+ by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin- tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca2+, reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 Å for Cys-96 and 35.2 Å for Cys-117. In the presence of Ca2+, Cys-96 moved away from actin Cys-374 by ~3.6 Å, while Cys-117 moved away by ~8 Å. This suggests the existence of a flexible 'hinge' region near the middle of TnI, allowing amino acid residues in the N- terminal half of TnI to interact with TnC in a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.

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U2 - 10.1021/bi991903b

DO - 10.1021/bi991903b

M3 - Article

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AN - SCOPUS:0034635132

VL - 39

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EP - 91

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 1

ER -