Contributions from all over - Widely distributed residues in thymosin beta-4 affect the kinetics and stability of actin binding

We have used site-directed mutagenesis to investigate the contributions of widely distributed residues in the thymosin beta-4 (T beta 4) sequence to the formation and stability of the actin-T beta 4 complex. Equilibrium and kinetic studies of actin binding were performed by monitoring the change in fluorescence of an N-iodoacetyl-N9-(5-sulfo-1-naphthyl)ethylenediamine (Aedans) fluorophore on actin cysteine-374. We evaluated the contributions of hydrophobic residues throughout T beta 4, the conserved and variable proline residues, and the conserved lysine residues to the kinetics and thermodynamics of T beta 4 binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted by alanine residues. All these mutations weaken the affinity of the actin-T beta 4 complex, but the kinetic basis of the lower stability of the complex varies among the mutants. Our results support a model in which T beta 4 initially binds actin through an electrostatic interaction, followed by the formation of widely distributed hydrophobic contacts. Several mutants, particularly at proline residues, dissociate much more rapidly than the wild-type complex, but also show small increases in the rate of association. This seeming paradox suggests that conformational searching of T beta 4, and particularly cis-trans isomerization of proline residues, contributes to the slow association rate constant of T beta 4, and to the stability of the hydrophobic contacts associated with strong actin binding.