FIGURE 1.

Ca²⁺ dissociation from IANBD-labeled TnC in Tn and Tn-exchanged rabbit rigor myofibrils. Panel A shows the time course of IANBD fluorescence as Ca²⁺ was chelated by EGTA and removed from the regulatory binding site of TnC_IANBD^T53C reconstituted into Tn. The Tn (0.3 μm) in Buffer A + 200 μm Ca²⁺ was rapidly mixed with an equal volume of Buffer A + 10 mm EGTA at 15 °C (Ca²⁺ off Tn trace, 40.8 ± 0.5/s). The Ca²⁺-saturated Tn base line was collected by mixing the Ca²⁺-saturated Tn with Buffer A + 200 μm Ca²⁺. The Ca²⁺-free Tn base line was acquired by rapidly mixing Tn in Buffer A + 5 mm EGTA against equal volumes of Buffer A + 5 mm EGTA. Panel B shows the time course of the IANBD fluorescence as Ca²⁺ was removed by EGTA from TnC_IANBD^T53C Tn-exchanged rabbit rigor ventricular myofibrils. TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ were rapidly mixed with an equal volume of the Buffer A + 10 mm EGTA at 15 °C (Ca²⁺ off Myofibrils trace, 25.3 ± 0.7/s). The Ca²⁺-saturated and Ca²⁺-free myofibril base lines were acquired from TnC_IANBD^T53C myofibrils under same buffer conditions as described for TnC_IANBD^T53CTn in panel A.