The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers

J. M. Francois, Z. Sheng, Danuta Szczesna-Cordary, J. D. Potter

Research output: Contribution to journalArticle

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Abstract

Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1- 100) sites I and II; and TH4 (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH1 was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH3 and TH4) or 20% (TH2) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH2, TH3, and TH4 compared to RSTnC or TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH1 was able to interact with the fibers in a Ca2+-independent (Mg2+-dependent) manner, indicating that at a minimum, Ca2+-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca2+. TH2 and TH4 appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH2 and TH4 was abolished when Ca2+ was removed. TH2 and TH4 binding to the second site required Ca2+ initially but was maintained in the presence of Mg2+. This interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca2+-Mg2+ sites (III and IV) of TnC.

Original languageEnglish
Pages (from-to)19287-19293
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number33
DOIs
StatePublished - Jan 1 1995

Fingerprint

Troponin C
Thrombin
Muscle
Rabbits
Muscles
Fibers
Binding Sites
Restoration
Proteolysis
Skeletal Muscle Fibers
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers. / Francois, J. M.; Sheng, Z.; Szczesna-Cordary, Danuta; Potter, J. D.

In: Journal of Biological Chemistry, Vol. 270, No. 33, 01.01.1995, p. 19287-19293.

Research output: Contribution to journalArticle

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abstract = "Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1- 100) sites I and II; and TH4 (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH1 was the most effective in the force restoration (65{\%}) whereas the smaller fragments developed about 50{\%} (TH3 and TH4) or 20{\%} (TH2) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH2, TH3, and TH4 compared to RSTnC or TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH1 was able to interact with the fibers in a Ca2+-independent (Mg2+-dependent) manner, indicating that at a minimum, Ca2+-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca2+. TH2 and TH4 appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH2 and TH4 was abolished when Ca2+ was removed. TH2 and TH4 binding to the second site required Ca2+ initially but was maintained in the presence of Mg2+. This interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca2+-Mg2+ sites (III and IV) of TnC.",
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T1 - The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers

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AU - Potter, J. D.

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N2 - Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1- 100) sites I and II; and TH4 (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH1 was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH3 and TH4) or 20% (TH2) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH2, TH3, and TH4 compared to RSTnC or TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH1 was able to interact with the fibers in a Ca2+-independent (Mg2+-dependent) manner, indicating that at a minimum, Ca2+-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca2+. TH2 and TH4 appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH2 and TH4 was abolished when Ca2+ was removed. TH2 and TH4 binding to the second site required Ca2+ initially but was maintained in the presence of Mg2+. This interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca2+-Mg2+ sites (III and IV) of TnC.

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