Conformational Modulation of Troponin T by Configuration of the NH₂-Terminal Variable Region and Functional Effects

Abstract

Troponin T (TnT) is an essential element in the thin filament-based regulatory system of striated muscle. Alternative mRNA splicing generates multiple TnT isoforms with primary structural differences in the NH2-terminal region. The functional significance of this hypervariable NH2-terminal domain and the developmental or muscle type-specific TnT isoforms is not fully understood. We have analyzed chicken breast muscle TnT containing a metal-binding cluster [H(E/A)EAH]4-7 (Tx) in the NH2-terminal region to demonstrate potential effects of the NH2-terminal structure on the conformation of TnT [Ogut, O., and Jin, J.-P. (1996) Biochemistry 35, 16581-16590]. Using specific antibody epitope analysis on this metal-binding TnT model, this study revealed that the binding of Zn2+ to the NH2-terminal region of chicken breast muscle TnT induces extensive conformational changes in the whole protein as demonstrated by a significant decrease in binding avidity of a polyclonal anti-TnT serum which recognizes multiple epitopes on the TnT molecule. This NH2-terminal configuration-based effect is not restricted to the metal ion interaction, whereas the binding of anti-NH2 terminus monoclonal antibodies to TnT induced similar changes. Protein-binding assays have shown that the NH2-terminal variability-induced conformational changes can alter TnT's binding affinity for tropomyosin and troponin I. The results suggest a functional modulation of TnT through the configuration of the NH2-terminal domain, and this novel mechanism may mediate the physiological significance of the TnT isoform regulation.

A role for serine-175 in modulating the molecular conformation of calponin

“…We have successfully applied this approach for structurefunction analysis of troponin T [41,45]. The epitope analysis Serine-175-based conformational modulation of calponin mAb epitope analysis clearly showed that the molecular conformation of calponin is reconfigured by phosphorylation and S175A substitution, supporting the regulatory function of serine-175.…”

“…Similar to the procedures which we have developed for studying troponin T isoforms [41], ELISA-based actin-calponin and tropomyosin-calponin binding assays were developed to characterize the interactions of calponin with the actin thin filament. As illustrated in Figure 2(C), microtitre plates were coated at 4 mC overnight with purified chicken gizzard actin and tropomyosin (alone or together at a molar ratio of 7 : 2, corresponding to the association of seven actins to a tropomyosin dimer) in buffer A.…”

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

“…Bovine cardiac TnI purified from ventricular muscle [10] and mouse cardiac/slow TnC purified from bacterial culture [10] were used as controls. The resulting gels were stained with Coomassie Brilliant Blue R250 to reveal the resolved protein bands and duplicate gels were electrically blotted to nitrocellulose membranes as previously described [18]. After blocking in 1% bovine serum albumin, the nitrocellulose membranes were incubated with an anti-cardiac TnI monoclonal antibody (mAb) at 4°C overnight.…”

“…After blocking in 1% bovine serum albumin, the nitrocellulose membranes were incubated with an anti-cardiac TnI monoclonal antibody (mAb) TnI-1 [19] at 4°C overnight. The membranes were then repetitively washed under high stringency using Tris-buffered saline containing 0.5% Triton X-100 and 0.05% SDS, incubated with alkaline phosphatase-labeled anti-mouse IgG second antibody (Sigma Chemical Co.) at room temperature for 1 hour, washed again as above, and developed in 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium substrate solution as previously described [18].…”

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl

“…The different isoforms also show differing actomyosin ATPase activity within the sarcomeres. Exclusion of the 10 amino acids in adult cTnT, encoded by exon 5, results in significant changes in activation of the actomyosin ATPase when compared to the embryonic isoforms [8,9]; not surprisingly, much smaller functional differences are observed from alternate splicing of exon 4 in bovine cardiac troponin T [10].…”