TROPONIN-T AND CA2-I IN THE TERNARY TROPONIN COMPLEX AND RECONSTITUTED THIN-FILAMENTS( DEPENDENCE OF THE DISTANCE BETWEEN CYS48 AND CYS133 OF TROPONIN)

Contraction of vertebrate striated muscle is regulated by the interact ion of Ca2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although mu ch is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibit ory subunit, responds to the binding of Ca2+ to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N- and C-terminal domains, respect ively, of TnI. A mutant rabbit skeletal TnI, TnI(48/133) (C64S), was c onstructed by converting Cys64 into Ser. The remaining two thiols at C ys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, a nd the nonfluorescent acceptor, DAB-Mal. We found an interprobe distan ce of similar to 41 Angstrom for both uncomplexed TnI and TnI in the b inary complex with TnC. This distance increased to 51 Angstrom in the ternary Tn complex with TnT. These distances did not change significan tly on binding of Ca2+ to TnC. In the reconstituted thin filament, thi s distance remained to be 50 Angstrom in the presence of saturating Ca 2+, but increased to similar to 66 Angstrom on removing Ca2+ with EGTA in the presence of Mg2+. Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca2+-dependent chang e in the global conformation of TnI in the Tn complex free in solution , the removal of Ca2+ caused a substantial separation of the N- and C- terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the rela xed state.