Disulfide Cross-Linked Antiparallel Actin Dimer

Abstract

Oxidation of actin monomer (G-actin) with copper o-phenanthroline resulted in a rapid, high yield of disulfide cross-linked dimer. The cross-link is due to an intermolecular disulfide bond between actin Cys374 of each molecule, resulting in a tail-to-tail, i.e., antiparallel, actin dimer. Analytical ultracentrifugation profiles of G-actin can be ascribed to the existence of actin monomers with very little, if any, dimer. Thus, actin dimers are not energetically favorable, indicating that cross-linked dimers are formed during random diffusional collisions. On the other hand, a similar oxidation of actin polymer (F-actin) resulted in a much lower yield of the cross-linked actin dimer that showed no sign of leveling off. Therefore, it is proposed that the cross-linked dimer from actin polymer is due to collisional complexes of actin monomers that are in equilibrium with the polymer during actin treadmilling. These results account for the reported observation that during the early stages of actin polymerization (where the actin monomer concentration is high) cross-linked antiparallel actin dimers are formed in relatively high yield whereas none are formed at later stages of polymerization. These findings raise questions concerning the validity of the antiparallel actin dimer model of in vitro actin polymerization that is based on the assumption that the ability to form cross-linked actin dimers implies the existence of stable dimers.