Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C

Abstract

A detailed mechanism that fully accounts for the Mg2+-induced polymerization of actin in the presence or absence of Ca2+ at 20 degrees C and pH 8 is presented. In the absence of Ca2+, the mechanism of the Mg2+-induced polymerization is as follows: Mg2+ binds to a metal-binding site on G-actin and induces a conformational change, which is required for eventual polymerization. The overall dissociation constant for this binding is about 30 microM. This actin species then binds a second molecule of Mg2+ (Kd = 5 mM), which yields a species capable of polymerization. Dimer formation from this monomeric species is quite unfavorable, but trimer formation from dimer and monomer is much more favorable. The trimer may then elongate to give filaments. Ca2+, when present, binds at the same site as the tightly bound Mg2+ and must be displaced by Mg2+ before the conformational change can occur. The rate and dissociation constants for tight binding of Ca2+ and Mg2+ and for the conformational change are consistent with those observed previously by using a fluorescently labeled G-actin. With the mechanism proposed, it is possible to fit the full time course of polymerization over a wide range of actin concentrations, Mg2+ concentrations, and Ca2+ concentrations.