The excimer fluorescence of pyrene-labeled tropomyosin. A probe of conformational dynamics

Abstract

Rabbit skeletal and cardiac tropomyosin were specifically labeled at their cysteine side chains with N-(1-pyrene)-maleimide. A high degree of intramolecular excimer formation due to interaction between adjacent pyrenes on Cys 190 of each chain was observed by fluorescence techniques. This modification produced low values of specific viscosity at low salt, indicating an inhibition of the salt-dependent polymerizability. Despite the loss of polymerizability, the excimer fluorescence of pyrene-tropomyosin increased with a similar salt dependence as the decrease in viscosity of an unlabeled control. Transient and steady state fluorescence measurements on the labeled tropomyosin indicated the presence of two states of labeled tropomyosin, an excimer-forming state (State B) and a nonexcimer-forming state (State A), in equilibrium with each other. The increase in excimer with increasing temperature and salt concentration can be explained by a shift in equilibrium toward State B. Steric considerations suggest that, in order for the pyrenes to form an excimer, localized chain separation in State B is required.