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Thymosin ß₄ Induces a Conformational Change in Actin Monomers

Irina V. Dedova ^*, Olga P. Nikolaeva , Daniel Safer , Enrique M. De La Cruz  and Cris G. dos Remedios ^*

^* Muscle Research Unit, Institute for Biomedical Research, University of Sydney, New South Wales, Australia; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia; Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania; and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut

Correspondence: Address reprint requests to Enrique M. De La Cruz, Yale University, Dept. of Molecular Biophysics and Biochemistry, PO Box 208114, New Haven, CT 06520-8114. Tel.: 203-432-5424; Fax 203-432-1296; E-mail: enrique.delacruz{at}yale.edu.

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin ß₄ (tß₄) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tß₄ 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 (ATP) and Lys-61 by 1.9 Å, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tß₄ binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tß₄ binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tß₄-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. Tß₄ and cofilin compete for actin binding. Tß₄ 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 tß₄-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tß₄-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.