Regulation of Muscle Contraction: Site-Directed Mutants of Thin Filament Components

• Grant number: 95/04527-3
• Support Opportunities: Research Projects - Thematic Grants
• Duration: May 01, 1996 - August 31, 2001
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Fernando de Castro Reinach
• Grantee: Fernando de Castro Reinach
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The contraction of both skeletal and cardiac muscles is regulated by the intracellular free calcium concentration. A molecular switch capable of detecting fluctuations in calcium levels regulates the interactions between actin and myosin, the proteins responsible for force generation, and therefore muscle contraction. The existence of this switch, the general principles of its operation and the proteins involved have been established over the past two decades. Our goal has been to define the detailed mechanisms of its operation at the molecular level. The switch consists of two proteins and a total of four polypeptides stably assembled on the surface of the actin filaments. The first protein, troponin, contains three polypeptides: calcium binding component (TnC, troponin C) an inhibitory component (TnI, troponin I) and a component that binds tropomyosin (TnT, troponin T). The second protein, tropomyosin is a coiled-coil dimer that interacts with one troponin molecule and seven actin monomers along the filament. The stoichiometry of the system is such that one troponin molecule regulates the interaction with myosin of seven actin monomers. An attractive feature of this system is that a fully functional troponin/tropomyosin complex can be assembled from the denatured subunits. This allows the incorporation of site-directed mutants produced in E. Coli into the complex. We have cloned and expressed the isolated components and the full troponin complex in E.coli. Using these systems we have introduced mutations and deletions in all components of the system and reconstituted muscle fibers and actin filaments with mutants. These results led us to understand the calcium induced conformational change of TnC and the relationships of the different subunits within the complex. We are now mapping the regions involved in the calcium induced conformational change in each component of the system. For that purpose we developed a method capable of incorporating tryptophan (trp) analogues with different spectral emission properties into recombinant proteins. (AU)