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Abstract

Binding sites of actin and thymosin β4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys, Cys, or to the sulfur atom of the ATP analog adenosine 5`-O-(thiotriphosphate) (ATPS), the actin derivatives were reacted with synthetic thymosin β4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys of actin and Cys of thymosin β4, indicating that the N terminus of thymosin β4 is in close proximity (≤9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin β4 analogs when the cross-linkers were anchored at Cys of actin. A second contact site was identified by cross-linking of Cys and Cys in thymosin β4 with the ATPS derivative bound to actin, indicating that the hexamotif of thymosin β4 (positions 17-22) is in close proximity (≤9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin β4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization