Biophysical studies on the calcium trigger of muscle contraction.

Abstract

The basic structural organization of striated muscle tissue consists of two types of filaments—thick and thin—which are interdigitated and slide past one another when muscle contracts. In the thick filaments the long matchsticklike myosin molecules are organized into a biopolar structure in which the tails form the core of the filament and the heads project out at both ends. The ATPase activity and acitin-binding properties are located in the projecting heads. It is the interaction of these heads with the thin filaments and the concomitant hydrolysis of ATP that provides the driving force for the sliding of thin over thick filaments. The core of the thin-filament structure is made up of a long double-stranded helical assembly of globular (G) acting monomers known as “F-actin.” Located in each of the two grooves of the F-actin structure is a filament of rodlike tropomyosin molecules aggregated head to tail and spanning the length of the thin filament. Each tropomyosin molecule spans seven G-actin monomers on each of the two strands of F-actin and interacts with one troponin complex. The latter consists of three proteins: troponin-C (TN-C), which binds calcium; troponin-I (TN-I), the inhibitory protein; and troponin-T (TN-T), which binds the troponin complex to tropomyosin. When a nerve impulse stimulates a muscle to contract there is an increase in the Ca2+ concentration in the fluid bathing the thick and thin filaments. Binding of this Ca2+ to the TN-C component triggers a series of conformational transitions in the troponin complex and a change in the position of tropomyosin from a blocking position in the grooves of F-actin. Myosin heads are then able to interact with F-actin and sliding of thick over thin filaments ensues. The research in our laboratory has been directed toward a fuller understanding of the detailed molecular mechanisms by which this intricate and sophisticated system operates in both skeletal and cardiac muscle. To this end we have explored the calcium-induced conformational change in TN-C and its propagation through the entire troponin–tropomyosin complex, using a combination of hydrodynamic (ultracentrifuge, viscosity, magnetic densitometry) and spectroscopic (CD, UV absorption difference, solvent perturbation difference, nmr, and fluorescence) approaches. Our studies to date have included the following: the development of highly purified troponin subunits, using high-performance liquid chromatography methodologies; the rigorous physicochemical characterization of the troponin subunits and their interaction properties; precise details of several of the functional groups (carboxyls, aromatics) involved in the Ca2+-induced conformational change in TN-C; the role of sulfhydryl groups in generating functionally active and conformationally sensitive troponin complexes; the molecular morphology of the troponin complex on the thin filament; the role of the two Ca2+-specific regulatory sites in TN-C in terms of their responsiveness to rapid Ca2+ transients; and the demonstration of a common regulatory mechanism for both skeletal and cardiac muscle, using bioassay and spectroscopic studies on ternary complexes made from hybrid subunits of both muscle types. The highlights of these studies are described, including the recently elucidated x-ray structure of TN-C and how the binding of calcium to such a structure may act as a conformational switch.