Structurally Unstable Regions in the Tropomyosin-Troponin Complex from Bovine Heart Muscle

Abstract

Regulation of contraction in striated muscles requires a semi-independent movement of various domains of the tropomyosin-troponin (TmTn) complex on the actin filament. Such movement is facilitated by the presence of flexible or structurally unstable regions in these proteins. In particular, it is well documented that the central part of tropomyosin comprises a region that unfolds readily at physiological temperatures. To identify flexible regions in the native TmTn complex isolated from bovine heart muscle, we have used limited proteolysis with Staphylococcus aureus V8 protease that cleaves specifically the peptide bond on the C-terminal side of Glu. At 4°C only troponin T and troponin I are digested at positions 59 and 166, respectively. At 35°C Tm is also digested, initially at position 145, followed by a rapid digestion at positions 142,131 and 150. The resulting two fragments of Tm spanning residues 1-131 (2x14.9 kDa) and 151-284 (2x15.5 kDa) are resistant to further digestion in the presence of troponin, which indicates that they retain their coiled-coil structure. However, in the absence of Tn the C-terminal fragment of Tm undergoes further proteolysis. The troponin C component of the complex is resistant to proteolysis under the conditions used. The two Tm fragments, together with TnC, the N-terminal fragment of TnI (residues 1-166) and the C-terminal fragment of TnT (residues 60-284) form a 125 kDa complex that is stable at low salt concentrations, binds to F-actin irrespective of calcium, and imparts significant calcium-dependent regulation to actomyosin ATPase. These results demonstrate that the central segment of Tm spanning residues 131-151 is structurally unstable irrespective of the presence of troponin and it structure stabilizing effect on the C-terminal part of Tm.