Open AccessStructural and Functional Domains of the Troponin Complex Revealed by Limited Digestion
Author(s) -
Takeda Soichi,
Kobayashi Tomoyoshi,
Taniguchi Hisaaki,
Hayashi Hiroshi,
Maéda Yuichiro
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00611.x
Subject(s) - proteolysis , peptide , chemistry , egta , troponin c , biochemistry , biophysics , atpase , calcium , troponin i , enzyme , biology , psychology , organic chemistry , psychiatry , myocardial infarction
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 Ca 2+ concentration, the cleavage sites of TnI were dependent on the Ca 2+ 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–TnI Ca‐frag complex [TnI Ca‐frag = (TnI–(1–134 and 1–140)‐peptide], which was produced in the presence of CaCl 2 and MgCl 2 , retains both the activating and inhibitory capabilities of whole Tn on the acto‐S1 ATPase activity, while TnT‐(159–259)‐peptide–TnC–TnI Mg‐frag complex [TnI Mg‐frag = (TnI‐(1–116)‐peptide], which was obtained in the presence of MgCl 2 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 tropomyosinactin filament.