Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

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• 作者：Barbara Spolaore ; Samanta Raboni ; Amparo Ramos Molina ; Abhijeet Satwekar ; Nunzio Damiano ; Angelo Fontana
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 30, 2012
• 年：2012
• 卷：51
• 期：43
• 页码：8679-8689
• 全文大小：497K
• 年卷期：v.51,no.43(October 30, 2012)
• ISSN：1520-4995

文摘

The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH₂ + H₂N-B 鈫?A-CONH-B + NH₃ reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), 伪-lactalbumin (伪-LA), and fragment 205鈥?16 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the 鈥渉elix F鈥?region in apoMb, in the 尾-domain in apo-伪-LA in its molten globule state, and in the N-terminal region in fragment 205鈥?16 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.