The Highly Conserved COOH Terminus of Troponin I Forms a Ca²⁺-Modulated Allosteric Domain in the Troponin Complex

Abstract

The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.

Localization and function of Xinα in mouse skeletal muscle

“…The membranes were then incubated with mAb T12 recognizing fast skeletal muscle troponin T (TnT; Ref. 36) and mAb TnI-1 recognizing all three muscle type TnI isoforms (25), both diluted in TBS containing 0.1% BSA, at 4°C overnight. After high stringency washes with TBS plus 0.5% Triton X-100 and 0.05% SDS, the membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG second antibody (Santa Cruz Biotechnology), washed again, and developed in 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium substrate solution.…”

Toad Heart Utilizes Exclusively Slow Skeletal Muscle Troponin T

“…The expression and purification of human cardiac TnT, immunization of Balb/c mice, hybridoma fusion, screening, and subcloning, mAb production, and immunoglobulin isotyping were carried out as described previously using standard methods (6). Isoform specificity of the anti-TnT mAbs obtained was examined with Western blots on cardiac and skeletal muscle samples from multiple vertebrate species.…”

Coexistence of cardiac troponin T variants reduces heart efficiency

“…The protein bands resolved were visualized with Coomassie blue R250 staining or electrically transferred to a nitrocellulose membrane using a semidry apparatus (Bio-Rad). Blotted membranes were blocked with Tris-buffered saline (TBS) containing 1% BSA and probed with anti-cTnT monoclonal antibody CT3 (22) or anti-TnI monoclonal antibody TnI-1 (24). The following washes and incubation with alkaline phosphatase-labeled anti-mouse IgG secondary antibody (Santa Cruz Biotechnology) and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium substrate reaction were carried out as previously described (9).…”