A fluorescent probe for conformational changes in skeletal muscle G-actin

Abstract

Actin from rabbit skeletal muscle has been modified with the fluorescent label N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS). Under conditions where the actin is in the unpolymerized form (G-actin), the addition of Mg2+ or KCl results in enhancement of the fluorescence. Titration of the labeled G-actin with Mg2+ at varying concentrations of CaCl2 gives, by extrapolation, a value for the dissociation constant for Mg2+ of 35 microM in the absence of Ca2+ and a calculated value of 10 microM for Ca2+ in the absence of Mg2+. The two metal ions compete with each other. The fluorescence enhancement induced by Mg2+ is reversed by the addition of Ca2+ and both processes are time-dependent, indicating a reversible conformational change of G-actin as a consequence of addition of divalent metal. KCl also enhances the fluorescence of the labeled G-actin but does not appear to compete with the divalent metal ion. The enhancement of the fluorescence is very rapid and any conformational change induced by KCl is probably different from that induced by divalent metal ions. Finally, it is shown that loss of fluorescence of the labeled G-actin may be associated with inactivation of the actin.