Contraction in striated muscle is regulated through a cascade of protein-protein interactions initiated by the binding of Ca2+-to cardiac troponin C (cTnC), the regulatory subunit of the troponin complex. The regulatory properties of cardiac and fast skeletal muscle TnC are more dynamic than a simple on/off molecular switch, and may be modulated by a variety of factors including phosphorylation of other regulatory proteins and degree of myosin crossbridges. The critical role for cTnC in muscle contraction emphasizes a need to understand its molecular mechanism of action. The goal of this proposal is to study structure/function relationships in cTnC using a synergistic blend of NMR spectroscopy and molecular biology to elucidate Ca2+-dependent protein dynamics essential for triggering and modulating muscle contraction. The general strategy will be to use a bacterial expression system to mutate selected amino acids in cTnC and enrich the normal and mutated proteins with amino acids labeled with stable isotopes to permit easy detection by NMR. This will allow the analysis of the whole protein, and interactions between cTnC and other regulatory proteins. A detailed understanding of structure/function relationships in cTnC may provide insights into how muscle contraction may be artificially modified by designing reagents that specifically interfere with the normal function of cTnC. Moreover, molecular mechanisms of action identified for TnC may be applicable to other Ca2+-binding proteins with activator activity such as calmodulin. The first Specific Aim will be to systematically mutate Met and Cys residues and label the mutated protein with [methyl-13C]Met. Assigned Met residues will be used as markers for conformational changes. Mono-Cys derivatives of cTnC will be used for site-specific attachment of fluorescent and spin-label probes. For the second Specific Aim, NMR will be used to monitor conformational changes in labeled proteins. Protein dynamics will be followed as a function of Ca2+ and Mg2+ binding; association with TnI, TnT and TnI peptides; phosphorylation of TnI and TnI peptides; and pH. The third Specific Aim will be to make sequential assignments and define the partial solution structure of cTnC.
Showing the most recent 10 out of 14 publications
