NMR studies of the calcium-induced structural changes that trigger muscle contraction

Skeletal and cardiac muscle contraction is triggered by the binding of calcium ions to the muscle thin-filament protein troponin C (TnC). The resulting conformational change alters the interaction of TnC with other proteins in the thin filament leading to muscle contraction. We have used multinuclear, multidimensional, high-resolution nuclear magnetic resonance (NMR) spectroscopic techniques to study this calcium-induced conformational change in TnC. This paper discusses the problems that exist with respect to NMR structural approaches in complex biological systems, i.e., the size limit and the aggregation of the protein, and the strategies we have taken to deal with these problems. These include the expression of only the regulatory domain of the protein and finding unique solution conditions for the intact protein. TnC undergoes a calcium-induced dimerization at neutral pH. Our studies indicate that the mode of dimerization involves the N-domain of one monomer interacting with the N-domain of another monomer. Addition of the solvent trifluoroethanol breaks the dimer effectively, making TnC predominantly a monomer. Under this condition, we were able to study the C-13, N-15-labeled TnC protein using NMR and to determine its structure in solution. The structure shows that the two domains are connected by a flexible linker. We have cloned and expressed the regulatory N-domain of the protein TnC and solved the complete three-dimensional structure of this protein in both the apo- and calcium-saturated forms using the C-13- and N-15-labeled proteins. These structures provide a clear description of the calcium-induced structural change. A simple method for characterizing the NMR properties of a given new protein to determine its chances of success in subsequent NMR experiments is also discussed.