Modulation of troponin T molecular conformation and flexibility by metal ion binding to the NH2-terminal variable region.

Abstract

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.