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 language | English |
|---|---|
| Pages (from-to) | 86-91 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 39 |
| Issue number | 1 |
| DOIs | |
| State | Published - 11 Jan 2000 |
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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 journal › Article
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.
UR - http://www.scopus.com/inward/record.url?scp=0034635132&partnerID=8YFLogxK
U2 - 10.1021/bi991903b
DO - 10.1021/bi991903b
M3 - Article
C2 - 10625482
AN - SCOPUS:0034635132
VL - 39
SP - 86
EP - 91
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 1
ER -