Cofilin and DNase I affect the conformation of the small domain of actin.

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 A (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 A), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 A) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells

Thymosin β(4) Induces a Conformational Change in Actin Monomers

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin β(4) (tβ(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tβ(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 Å and decreases the distance between the purine base of bound ATP (ɛATP) and Lys-61 by 1.9 Å, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tβ(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tβ(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tβ(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tβ(4) and cofilin compete for actin binding. Tβ(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tβ(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tβ(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding