Formation and implications of a ternary complex of profilin, thymosin beta(4), and actin

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin be ta (4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta (4) (residues 6-22) confirm the presence of an extensive bind ing surface between actin and thymosin beta (4), and explain why thymosin b eta (4) and profilin can bind simultaneously to actin. Surprisingly, despit e much lower affinity, the N-terminal thymosin beta (4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurrin g at the N terminus of thymosin beta (4). Intracellular concentrations of t hymosin beta (4) and profilin may greatly exceed the equilibrium dissociati on constant of the ternary complex, inconsistent with models showing sequen tial formation of complexes of profilin-actin or thymosin beta (4)-actin du ring dynamic remodeling of the actin cytoskeleton. The formation of a terna ry complex results in a very large amplification mechanism by which profili n and thymosin beta (4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant seque stration even if molecular crowding results in a very low critical concentr ation of actin in vivo.