High-resolution HPLC-ESI-MS characterization of the contact sites of the actin-thymosin ß(4) complex by chemical and enzymatic cross-linking.
Biochemistry
; 52(33): 5553-62, 2013 Aug 20.
Article
em En
| MEDLINE
| ID: mdl-23924371
ABSTRACT
Thymosin ß4 sequesters actin by formation of a 11 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 ß4 was cross-linked to E167 of actin, K18 or K19 of thymosin ß4 to one of the first three amino acids of actin (DDE), and S43 of thymosin ß4 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 ß4 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 ß4, and K61 of actin to Q39 of thymosin ß4. The third cross-linking site was identified between Q41 of actin and Q39 of thymosin ß4, which are simultaneously cross-linked to K16, K18, or K19 of thymosin ß4. When both cross-linking reactions are taken together, the complex formation of actin by thymosin ß4 is more likely to be flexible than rigid and is localized along the subdomains 1 to 3 of actin.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Timosina
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Cromatografia Líquida de Alta Pressão
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Actinas
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Espectrometria de Massas por Ionização por Electrospray
Limite:
Animals
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article