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

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca2+-regulation of striated muscle contraction. Develop mentally regulated alternative RNA splicing produces TnT isoforms differing in their NH2-terminal structure. Physical property variations of the NH2-t erminal hypervariable region of TnT may have a role in tuning the Ca2+-sens itivity and overall cooperativity of the muscle. We have previously demonst rated that metal ion or monoclonal antibody binding to the NH2-terminal reg ion can modulate the epitopic conformation and troponin I and tropomyosin b inding affinity of TnT. To further establish the molecular basis of this co nformational and functional modulation, we have characterized the NH2-termi nal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isofo rm, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx ) in the NH2-terminal variable region was used in this study. TnT8e16 was t itrated for Cu(II) binding-induced changes in fluorescence intensity and an isotropy of the COOH-domain Trp residues (W234, W236, and W285), which demo nstrated considerable environmental sensitivity in TnT denaturation studies , Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion bindi ng-induced conformational change in TnT, Fluorescence anisotropy changes up on metal ion binding indicated a decrease in the mobility of the Trp residu es and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH2- terminal variable region of TnT modulates conformation and flexibility of o ther domains of the protein.