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Biophys. J. BioFAST: First Published November 4, 2005. doi:10.1529/biophysj.105.063081
© 2005 by the Biophysical Society.

A more recent version of this article appeared on February 1, 2006.
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MUSCLE AND CONTRACTILITY

Thymosin {beta}4 Induces a Conformational Change in Actin Monomers

Irina V Dedova 1, Olga P Nikolaeva 2, Daniel Safer 3, Enrique M De La Cruz 4* and Cris G dos Remedios 5

1 Muscle Research Unit, Institute for Biomedical Research, The University of Sydney
2 Belozersky Institute of Physico-Chemical Biology, Lomonosov State University, Moscow
3 Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania
4 Department of Molecular Biophysiscs and Biochemistry, Yale University
5 Muscle Research Unit, Institute for Biomedical Research, University of Sydney

* To whom correspondence should be addressed. E-mail: enrique.delacruz{at}yale.edu.

Submitted on March 16, 2005
Revised on May 2, 2005
Accepted on 17 October 2005


  Abstract
Using fluorescence resonance energy transfer (FRET) spectroscopy we demonstrate that thymosin [beta]4 (t[beta]4) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers. T[beta]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 ([epsilon]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 t[beta]4 binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry (DSC) t[beta]4 binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the t[beta]4-actin complex. Changes in FRET are accompanied by marked reduction in solvent accessibility of the probe at Gln-41 suggesting it forms part of the binding interface. T[beta]4 and cofilin compete for actin binding. T[beta]4 concentrations that compete cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high affinity t[beta]4 binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The t[beta]4-induced conformational change presumably accounts for the reduced rate of nucleotide and amide hydrogen exchange from actin monomers.

Key Words: FRET spectroscopy, actin, cofilin, differential scanning calorimetry, native PAGE gel electrophoresis, thymosin [beta]4






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Copyright © 2005 by the Biophysical Society.