Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
Biochemistry. 2013 Aug 20;52(33):5553-62. doi: 10.1021/bi400664k. Epub 2013 Aug 8.
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.
胸腺肽 β4 通过形成 1:1 复合物来隔离肌动蛋白。在复合物中，这种瞬时结合通过使用交联剂 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和微生物转谷氨酰胺酶形成共价键来稳定。通过 SDS-PAGE 分离产物后，通过胰蛋白酶胶内消化和高分辨率 HPLC-ESI-MS 确定交联位点的定位。在用化学交联进行交联后，鉴定了三个交联片段，表明存在三个接触位点。由于在化学交联后同时检测到交联片段和相应的非交联片段，因此这三个接触位点不是平行形成的。胸腺肽 β4 的 K3 与肌动蛋白的 E167 交联，胸腺肽 β4 的 K18 或 K19 与肌动蛋白的前三个氨基酸之一（DDE）交联，胸腺肽 β4 的 S43 与肌动蛋白的 H40 交联。事实证明，组氨酸的咪唑环是碳二亚胺介导的交联的酰基受体。分子建模证明，胸腺肽 β4 沿肌动蛋白的亚结构域 1 至 3 呈伸展构象。使用微生物转谷氨酰胺酶进行酶交联导致形成三个交联位点。肌动蛋白的 Q41 与胸腺肽 β4 的 K19 交联，肌动蛋白的 K61 与胸腺肽 β4 的 Q39 交联。第三个交联位点位于肌动蛋白的 Q41 和胸腺肽 β4 的 Q39 之间，它们同时与胸腺肽 β4 的 K16、K18 或 K19 交联。当两个交联反应一起考虑时，胸腺肽 β4 与肌动蛋白的复合物形成更可能是灵活的而不是刚性的，并且定位在肌动蛋白的亚结构域 1 至 3 上。
