Ca2+ induces an extended conformation of the inhibitory region of troponinI in cardiac muscle troponin

The inhibitory region of troponin I (TnI) plays a central regulatory role i n the contraction and relaxation cycle of skeletal and cardiac muscle throu gh its Ca2+-dependent interaction with actin. Detailed structural informati on on the interface between TnC and this region of TnI has been long in dis pute. We have used fluorescence resonance energy transfer (FRET) to investi gate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled w ith IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in c TnI brackets the inhibitory region (residues 130-149), and the distance bet ween the two sites was found to be 19.4 Angstrom in free cTnI. This distanc e was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca2+ in cTnC. An increase of 9 Angstrom in the Trp129-Cys152 separation was observed upon s aturation of the Call regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended con formation is different from a recent model of the Ca2+-saturated skeletal T nI-TnC complex in which the inhibitory region is modeled as a beta -turn. T he observed Ca2+-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca2+ activation in cardiac muscle. (C) 2001 Academic P ress.