MODULATION OF TROPONIN-C BINDING TO TROPONIN-T BY CA2+, PROBED BY FLUORESCENCE

The effects of Ca2+ on the binding interaction between troponin-C (TnC ) and troponin-T (TnT) and between TnC and troponin-I (TnI) were studi ed in both the binary complexes and the presence of other subunits and tropomyosin (Tm). Rabbit skeletal TnC was labeled with 7-dimethyl-ami no-4-methyl-3-N-meleimido-coumarin (DACM). The fluorescence titration curve of the labeled TnC with Ca2+ showed a biphasic response; fluores cence was quenched (by 31%) initially but increased (by 64%) in the se cond phase. In the presence of CaCl2 (3 mM), titrating labeled TnC wit h TnT caused further fluorescence enhancement (32-34%) of DACM by TnT. In the absence of Ca2+, TnT bound weakly to TnC (K)a) < 10(3) M-1) wh ereas in the presence of Ca2+, the TnT-TnC binding affinity increased almost 1000 fold (K(a) = 0.42 x 10(6) M-1). The affinity constant of u nlabeled TnC for TnT is 10 - 15 times that of the labeled complex (K(a ) = (3.4 approximately 5.1) x 10(6) M-1); this Value was determined fr om the binding isotherm of a mixed system consisting of labeled and un labeled TnC at a fixed ratio upon titration with TnT. Adding Tm to the system weakened the binding of TnT to TnC by at least 50 - 60 percent . Adding TnI to DACM-TnC alone also enhanced the fluorescence (20-22%) . An affinity constant 10(8) M-1 for TnC-TnI binary complex was obtain ed. If Tn-I and the labeled TnC were premixed first in 1:1 stoichiomet ry, then titrated with TnT, a further fluorescence increase (34%) simi lar to that in the absence of TnI was observed. The fit of the binding curve shows that K(a) of TnT to TnC increased (1.5 x 10(6) M-1). When TnI was added to the TnC-TnT complex at the end of titration when the fluorescence binding curve became leveled, quenching (12%) occurred. The latter result indicates that TnI competes with TnT for the same bi nding sites on TnC. As binding of TnC is at least 100 times as strong to TnI as to TnT, a quenching effect is observed. Furthermore, the con formation of TnC in the TnC-TnI binary complex may vary from that of T nC alone; binding of TnT to TnC is greatly enhanced (directly or indir ectly by TnI) in the TnC-TnI complex. These results indicate that the variation of binding affinity between TnC and TnT as modulated by Ca2 may play an important role in the Ca+2-regulation mechanism of muscle contraction.