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High-Resolution HPLC–ESI–MS Characterization of the Contact Sites of the Actin–Thymosin β4 Complex by Chemical and Enzymatic Cross-Linking

Knop, Jana, App, Christine, Horn, Anselm H. C., Iavarone, Federica, Castagnola, Massimo, Hannappel, Ewald
Biochemistry 2013 v.52 no.33 pp. 5553-5562
DDE (pesticide), actin, cross-linking reagents, crosslinking, histidine, molecular models, polyacrylamide gel electrophoresis, protein-glutamine gamma-glutamyltransferase, thymosin
Thymosin β₄ sequesters actin by formation of a 1:1 complex. This transient binding in the complex was stabilized by formation of covalent bonds using the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and a microbial transglutaminase. The localization of cross-linking sites was determined after separating the products using SDS-PAGE by tryptic in-gel digestion and high-resolution HPLC–ESI–MS. Three cross-linked fragments were identified after chemical cross-linking, indicating three contact sites. Because the cross-linked fragments were detected simultaneously with the corresponding non-cross-linked fragments, the three contact sites were not formed in parallel. K3 of thymosin β₄ was cross-linked to E167 of actin, K18 or K19 of thymosin β₄ to one of the first three amino acids of actin (DDE), and S43 of thymosin β₄ to H40 of actin. The imidazole ring of histidine was proven to be an acyl acceptor for carbodiimide-mediated cross-linking. Molecular modeling proved an extended conformation of thymosin β₄ along the subdomains 1 to 3 of actin. The enzymatic cross-linking using a microbial transglutaminase led to the formation of three cross-linking sites. Q41 of actin was cross-linked to K19 of thymosin β₄, and K61 of actin to Q39 of thymosin β₄. The third cross-linking site was identified between Q41 of actin and Q39 of thymosin β₄, which are simultaneously cross-linked to K16, K18, or K19 of thymosin β₄. When both cross-linking reactions are taken together, the complex formation of actin by thymosin β₄ is more likely to be flexible than rigid and is localized along the subdomains 1 to 3 of actin.