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 language | English |
|---|---|
| Pages (from-to) | 19287-19293 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 270 |
| Issue number | 33 |
| DOIs | |
| State | Published - Jan 1 1995 |
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ASJC Scopus subject areas
- Biochemistry
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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 journal › Article
}
TY - JOUR
T1 - The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers
AU - Francois, J. M.
AU - Sheng, Z.
AU - Szczesna-Cordary, Danuta
AU - Potter, J. D.
PY - 1995/1/1
Y1 - 1995/1/1
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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029121702&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029121702&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.33.19287
DO - 10.1074/jbc.270.33.19287
M3 - Article
C2 - 7642603
AN - SCOPUS:0029121702
VL - 270
SP - 19287
EP - 19293
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 33
ER -