Biophys J, May 2000, p. 2516-2527, Vol. 78, No. 5

Thymosin-₄ Changes the Conformation and Dynamics of Actin Monomers

Enrique M. De La Cruz,^* E. Michael Ostap,^* Rodney A. Brundage, K. S. Reddy, H. Lee Sweeney,^* and Daniel Safer^*

 ^*Pennsylvania Muscle Institute and Department of Physiology,  Department of Cell and Developmental Biology, and  Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 USA

Thymosin-₄ (T₄) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. T₄ binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys³⁷⁴ of actin monomers. The K_d of the actin-T₄ complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25°C, the free energy of T₄ binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly⁴⁶ on subdomain 2 is >12 times faster when T₄ is bound. The C terminus of T₄ contacts actin near this cleavage site, at His⁴⁰. By tritium exchange, T₄ slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that T₄ changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of T₄ to sequester actin monomers and inhibit nucleotide exchange.

Biophys J, May 2000, p. 2516-2527, Vol. 78, No. 5
© 2000 by the Biophysical Society   0006-3495/00/05/2516/12  $2.00

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