Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.is maintained at levels far above the critical concentration for polymerization (0.1 μM) (1) in specific areas of a mammalian cell. In lamellipodia, the levels of G-actin and filamentous actin (F-actin) are estimated to be 150 and 500 μM, respectively (2). Generally, actin filaments are thought to be capped to prevent nonproductive polymerization, whereas actively elongating filaments are created through controlled uncapping, severing, or de novo nucleation mechanisms (3) to harness the force of polymerization for a particular biological process. The availability of the pool of G-actin is tightly regulated through the binding of two classes of G-actin sequestering proteins (3, 4). β-Thymosins are short peptides (∼43 aa) that are able to completely sequester G-actin, preventing G-actin from forming a nucleus or joining either end of an existing actin filament. Profilins (molecular mass ∼ 16 kDa), in isolation, also sequester G-actin, precluding nucleation or participation in pointed-end filament elongation. In contrast, they actively participate in barbed-end filament elongation, dissociating from the actin protomer as it is incorporated into the filament. In the presence of actin nucleation and elongation machineries, such as actin-related protein 2/3 complex activators, formins, Wiskott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)-containing nucleators [Spire, protein Cordon-bleu (Cobl), and VopF/L], and vasodilator-stimulated phosphoprotein (VASP) (5, 6), profilin plays an active role in the nucleation and elongation processes.Cellular concentrations of thymosin-β4 (Tβ4) are in the 100-to 500-μM range, and its association with ATP-and ADP-bound actin is characterized by dissociation constant (K d ) values of 0.1-3.9 and 80-100 μM, respectively (7-11). Profilin I has been reported to be at a concentration of 8.4 μM in CHO cells (12) and can amount from 14% to 100% of the G-actin pool in other cells (13). Profilin I interacts with actin with K d values of 0.1 and 0.17 μM for ATP-an...