Fig. 7. Actin binding by the IRTKS Ct extension. (A) Alignment of IRTKS C-terminal extension with the WH2 domains of five human proteins. Residues conserved in at least three of the displayed WH2 domains are highlighted in black. Residues in IRTKS Ct conserved in the WH2 domains are shown in grey. Residues critical for actin monomer binding by thymosin 4 are indicated with asterisks. (B) Measurement of actin critical concentration by monitoring pyrene-actin fluorescence at a range of actin concentrations, alone or in the presence of 2.5 µM GST-Ct or GST-ScarWH2. Polymerisation of actin results in an increase in the gradient of fluorescence change with actin concentration and the critical concentration is derived from the intercept of the pre- and post-polymerisation gradients. Data shown are from three replicates of a single representative experiment. (C) Co-sedimentation assay at a range of F-actin concentrations showing binding of GST-Ct and GST-ScarWH2 to phalloidin stabilised F-actin. The quantification is described in the Materials and Methods. The binding curve fitted for GST-Ct gives a K_d of approximately 1 µM. GST-ScarWH2 data could not be fitted to a binding curve. Data shown are from three replicates of a single representative experiment.