Influence of the N Terminus and the Actin‐Binding Motif of Thymosin β 4 on Its Interaction with G‐Actin

:  Thymosin β 4 binds G‐actin in a 1:1 ratio and prevents its aggregation to F‐actin by sequestration. Substitution or modification of single amino acid residues within the N‐terminal sequence 1 to 22 of thymosin β 4 alters its interaction with G‐actin.We generated thymosin β 4 variants with amino acid substitutions within the N‐terminal α‐helix and the putative actin‐binding motif. None of the E. coli‐ generated thymosin β 4 variants was modified or acetylated at its N terminus. The stability of the complex of G‐actin with nonacetylated thymosin β 4 or β 4 A7V is higher than the one with naturally occurring thymosin β 4 , which is always acetylated. The complex of G‐actin with nonacetylated thymosin β 4 A7V,K18,19A and β 4 K14,16,18,19A is 15 times less stable compared to the complex with thymosin β 4 . The G‐actin sequestering activities of all thymosin β 4 variants correspond to their complex stabilities with G‐actin, except for nonacetylated thymosin β 4 A7V , where it is attenuated. Thymosin β 4 Δ17–23 missing the putative actin‐binding motif shows no interaction with G‐actin.