Two-site attachment of troponin to pyrene-labeled tropomyosin.
The interaction of troponin (a complex of troponin T, troponin I, and troponin C) with tropomyosin (Tm) was studied using the fluorescence of pyrene-labeled Tm. Fluorescence titrations showed that an increase in salt concentration not only decreased the binding constant of troponin to Tm but also decreased the magnitude of the total pyrene fluorescence change. This indicates that the interaction of the part of troponin (TnT2) with the middle of Tm near Cys-190 is weakened at high ionic strength, while the other part (TnT1) maintains its binding with the C-terminal region of Tm. The temperature dependence of the pyrene excimer fluorescence of Tm showed that troponin stabilized the whole Tm molecule with much less effect on the stability of the middle region where the TnT2 binds. TnT1, the N-terminal chymotryptic fragment of troponin T (TnT), stabilized Tm in a similar manner to troponin by inhibiting dissociation of Tm chains in the main unfolding transition. Thus, these data indicate a loose interaction of the TnT2 part of troponin with the region of Tm near Cys-190 and a tight interaction of the TnT1 part with the C-terminal region of Tm. TnT alone interacted more strongly with Tm near Cys-190 than when in the troponin complex. This was indicated by the following observations in the Tm.TnT complex compared to the Tm-troponin complex: (i) a more hydrophobic environment of pyrene at Cys-190 of Tm; (ii) stabilization against the local unfolding of Tm near Cys-190; (iii) more fluorescence resonance energy transfer between Tm and TnT. These studies indicate a loose and flexible interaction of troponin with the middle region of Tm which may be important in Ca2+ regulation by troponin.Tm.
Ishii
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Y
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Lehrer
S S
SS
D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D011721:Pyrenes; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*
D011721:Pyrenes; D014335:Tropomyosin; D014336:Troponin