Reversible dimer dissociation of tubulin S and tubulin detected by fluorescence anisotropy

Abstract

Concentration-dependent dissociation of dimers of goat brain tubulin S and tubulin was studied by fluorescence anisotropy. Upon dilution, assembly-competent fluorescein 5'-maleimide labeled dimers of tubulin S and tubulin show a progressive decrease in fluorescence anisotropy. That this lowering of anisotropy results from the dissociation of tubulin S dimers into monomers was shown by dilution experiments with unlabeled homologous and heterologous proteins. A nonlinear least-squares fit of the data gave a dissociation constant of 7.1×10⁻⁸ M for tubulin S compared to 7.2×10⁻⁷ M for tubulin at 25°C in 0.1 M PEM buffer, pH 7.0. van't Hoff plots of dimer-monomer dissociation of tubulin S and tubulin also show considerable differences in Δ H and Δ S. Effects of ionic strength and colchicine on the equilibrium constants are also substantially different for tubulin and tubulin S. The implications of these observations on the influence of C-terminal tails on tubulin structure are discussed.