CHARACTERISTICS OF TROPONIN-C BINDING TO THE MYOFIBRILLAR THIN FILAMENT - EXTRACTION OF TROPONIN-C IS NOT RANDOM ALONG THE LENGTH OF THE THIN FILAMENT

Troponin C (TnC) is the Ca2+-sensing subunit of troponin responsible f or initiating the cascade of events resulting in contraction of striat ed muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers, The properties of TnC extr action have not been characterized at the structural level, nor have t he interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunc tion with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomnes s of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained bi ological activity, as evidenced by reconstitution of Ca2+-dependent AT Pase activity in extracted myofibrils and binding to Tnl in a Ca2+-sen sitive manner. The binding of F5M TnC to highly extracted myofibrils a t low Ca2+ was restricted to the overlap region under rigor conditions , and the location of binding was not influenced by F5M TnC concentrat ion. The addition of myosin subfragment 1 to occupy all actin sites re sulted in F5M TnC being bound in both the overlap and nonoverlap regio ns. However, very little F5M TnC was bound to myofibrils under relaxin g conditions. These results suggest that strong binding of myosin head s enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding w as concentration dependent: binding was restricted to the overlap regi on at low F5M TnC concentration, whereas the binding propagated into t he nonoverlap region at higher levels. Analysis of fluorescence intens ity showed the greatest binding of F5M TnC at high Ca2+ with S1, and t hese conditions were used to characterize partially TnC-extracted myof ibrils. Comparison of partially extracted myofibrils showed that low l evels of extraction were associated with greater F5M TnC being bound i n the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not ra ndom along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the infl uence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2