Fluorometric Studies on Conformational Changes in Tropomyosin Associated with Depolymerization

Abstract

Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30°C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93×10^-5M (monomer) and 7.43×10^-5M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules.
Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.