Expression, zinc-affinity purification, and characterization of a novel metal-binding cluster in troponin T: metal-stabilized alpha-helical structure and effects of the NH2-terminal variable region on the conformation of intact troponin T and its association with tropomyosin.

A repeating metal-binding (Cu2+ > Ni2+ > Zn2+ approximately Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.-P., & Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2-terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn(2+)-affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated alpha-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn(2+)-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn(2+)-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca(2+)-activation of muscle contraction.