Skeletal Muscle Troponin C

Abstract

The use of skeletal muscle troponin C (TnC) in structural and functional studies ranging from characterization of its Ca²⁺-binding properties to interaction with troponin I (TnI) and troponin T (TnT), the other two members of the tripartite troponin regulatory complex, has necessitated the purification of large quantities of protein. The procedures for purification of the naturally occurring troponin components from muscle tissue, including preparation of a muscle ether powder, isolation of the crude troponin complex, and further fractionation using column chromatography to obtain the individual subunits, have been described in detail by Potter (1), and therefore, will not be discussed in this chapter. Owing to the problems with heterogeneity arising from copurification of different isoforms and/or variation in the extent of phosphorylation of the final isolated protein from muscle tissue, methodologies have since been developed using molecular biology strategies to express and purify homogeneous preparations of TnC, TnI, and TnT. Other advantages of these recombinant techniques include the ability of producing site-specific mutants and isolated fragments for studying specific regions without interference from the rest of the molecule, as well as ¹⁵N- and ¹³C-labeled derivatives for NMR studies.