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Troponin (Tn), consisting of three subunits, TnT, TnC, and TnI, plays a crucial
role in the calcium-dependent regulation of vertebrate striated muscle
contraction. In the present study, we have applied limited proteolysis to the Tn
complex in order to study domain structures and to detect conformational
differences of Tn under different conditions. We found that both TnT and TnI
were susceptible to chymotryptic digestion: while TnT was cleaved into
TnT-(1-158)-peptide and TnT-(159-259)-peptide irrespective of Ca2+
concentration, the cleavage sites of TnI were dependent on the Ca2+ occupancy of
TnC. In addition, we characterized the effects of depletion of the C-terminal
part of TnI on acto-S1 ATPase activity. The TnT-(159-259)-peptide-TnC-TnICa-frag
complex [TnICa-frag = (TnI-(1-134 and 1-140)-peptide], which was produced in the
presence of CaCl2 and MgCl2, retains both the activating and inhibitory
capabilities of whole Tn on the acto-S1 ATPase activity, while
TnT-(159-259)-peptide-TnC-TnIMg-frag complex [TnIMg-frag =
(TnI-(1-116)-peptide], which was obtained in the presence of MgCl2 and EGTA,
lost its ability to activate acto-S1 ATPase activity. Our results indicate that
residues 117-134 or 117-140 of TnI undergo structural changes upon
Ca(2+)-binding to the regulatory sites of TnC and are necessary for the
Ca(2+)-dependent inhibitory action of the Tn complex on acto-S1 ATPase activity.
We also showed that residues 135-181 or 141-181 of TnI are involved in the
interaction of Tn with the tropomyosin-actin filament.
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