Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments.
Contraction of vertebrate striated muscle is regulated by the interaction of Ca2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibitory 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, respectively, of TnI. A mutant rabbit skeletal TnI, TnI48/133 (C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of approximately 41 A for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 A in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca2+ to TnC. In the reconstituted thin filament, this distance remained to be 50 A in the presence of saturating Ca2+, but increased to approximately 66 A 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 change 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 relaxed state.
Luo
Y
Y
Muscle Research Group, Boston Biomedical Research Institute, 20 Staniford Street, Boston, Massachusetts 02114, USA. yinluo@bbri.harvard.edu
Wu
J L
JL
Gergely
J
J
Tao
T
T
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry*; D011817:Rabbits; D014336:Troponin / Q000737:chemistry*; D019210:Troponin I / Q000737:chemistry*; D020107:Troponin T
D014336:Troponin; D019210:Troponin I; D020107:Troponin T; D002118:Calcium