Conformation of Troponin and Myosin in Muscle Contraction

Unraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Since myosin is the motor of the muscle contractile system and troponin plays a key role in muscle regulation, this study is aimed at understanding conformational changes occurring in myosin and troponin during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with electron paramagnetic resonance (SDSL-EPR). Troponin is composed of three subunits: TnC, TnI and TnT. The structure of the inhibitory region of TnI in the troponin ternary complex was determined by measuring the accessibility of spin labels to a water-soluble spin relaxation agent. Results from SDSL-EPR scanning of residues 129 to 145 of cardiac TnI in the ternary complex (C.I.T) suggested that residues 129-137 region fold into an a-helix and residues 138-145 are unstructured. The ternary structure of TnC was determined by measuring distances between different domains of TnC using double electron electron resonance (DEER). We found that: (a) TnC in solution is flexible; (b) the calcium switch mechanism proposed for isolated TnC occurs in the troponin complex; (c) cardiac TnC central helix is collapsed in troponin ternary complex. Distance measurements between TnC and TnI revealed that there is no significant re-arrangement of the TnC/TnI interface in the TnC C-domain upon calcium binding; however, there is a large movement of the TnI switch peptide induced by binding of Ca2+. In the presence of calcium the switch peptide interacts with the TnC N-domain, while in the absence of Ca2+ the switch peptide moves closer to the TnI inhibitory region. A subfragment of the muscle motor protein, the myosin head, is alone capable of generating force and translation that results in muscle contraction. The myosin head has two distinct domains: a catalytic domain that binds actin and hydrolyzes ATP and a regulatory domain that functions as a lever. Hydrolysis of ATP and binding to actin induces strain within the upper and lower halves of the catalytic domains that is then amplified by the lever arm action of the regulatory domain. For this mechanism to be operative two things should hold: (a) the cleft between upper and lower domains of the catalytic domain closes on binding to actin and (b) the regulatory domain is able to move with respect to the catalytic domain. The first point, closure of the actin binding cleft upon binding of myosin to actin, was investigated using dipolar and pulsed-EPR experiments that are sensitive enough to to measure distances between spin labels placed on either site of the actin binding cleft. As hypothesized, the distances across the cleft were smaller in the presence of actin than in its absence. Movement of the regulatory domain was addressed by determining the domain dynamics of smooth muscle myosin using saturation transfer EPR and phosphorescence anisotropy. Results from these experiments revealed that the regulatory domain is 20-fold more restricted in motion than the catalytic domain implying that a hinge exists between the two domains. It is likely that the inhibition of the regulatory domain dynamics as compared to the skeletal muscle myosin correlates with the inhibited state of the smooth muscle myosin ATPase.