Expression, Zinc-Affinity Purification, and Characterization of a Novel Metal-Binding Cluster in Troponin T:  Metal-Stabilized α-Helical Structure and Effects of the NH₂-Terminal Variable Region on the Conformation of Intact Troponin T and Its Association with Tropomyosin

Abstract

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.

Identification of a Region of Troponin I Important in Signaling Cross-bridge-dependent Activation of Cardiac Myofilaments

“…ELISA and Binding Assay-To determine the affinity of cardiac TnT for the various TnIs, the ELISA-based solid-phase protein binding assay was carried out similar to that previously described (25,26). The concentration of Escherichia coli cell lysates containing GST-tagged fusion proteins of cardiac, slow skeletal, or mutant TnI required to completely saturate the well of Reacti-bind glutathione-coated plates (Pierce) was first determined by standard ELISA (27) carried out with the antiTnI specific monoclonal antibody C5 (Research Diagnostics Inc.).…”

Exon Skipping in Cardiac Troponin T of Turkeys with Inherited Dilated Cardiomyopathy

“…8 and Fig. 9) suggest that the deletion of exon 8 segment may have a profound effect on the function of cTnT based on the modulatory role of the NH 2 -terminal structure of TnT, which affects the conformation and function of other domains of the molecule (23,24,54). The proximal position of the exon 8-encoded segment to the Tm-binding site in the central region of TnT (42) (Fig.…”

Cooperative Interaction between Developmentally Regulated Troponin T and Tropomyosin Isoforms in the Absence of F-actin

“…For antibody incubations, TCEP was omitted. The bound mAbs were detected by horseradish peroxidase-conjugated anti-mouse immunoglobulin second antibody and the H 2 O 2 and 2,2Ј-azinobis-(3-ethylbenzthiazolinesulfonic acid) substrate reaction as described previously (28).…”