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• W51144307 abstract "Site-specific modification of proteins by transglutaminaseTransglutaminase (TGase; EC 2.3.2.13) catalyzes the reaction between the γ-amido group of a protein-bound Gln residue (–CONH2, the acceptor) and an amino group (–NH2, the donor) of an alkyl-amine (protein-CONH2 + H2N-ligand → protein-CONH-ligand + NH3). In the case of protein substrates, TGase causes an intra- and inter-molecular crosslinking of proteins by formation of an isopeptide bond involving the side chains of Gln and Lys residues. The acyl donor can be also a small amido-ligand mimicking the Gln residue, so that TGase allows a useful and interesting variability of substrates, thus leading to the modification of proteins at the level of Gln or Lys residues using appropriate substrate reagents.Recently, the microbial TGase from S. mobaraensis has attracted a strong interest for protein modification, considering its stability, high reactivity and small size. The X-ray structure of this TGase has been solved and shown to contain an active site given by a triad Cys-His-Asp in analogy to a protease. A striking result of recent studies is that reactions mediated by TGase can be site-specific with some proteins, sometimes leading to the modification of only one Gln residue among the many Gln residues of a protein substrate. On the other hand, there is only a moderate specificity for Lys residues. With the view to shed light into the molecular features dictating the site-specific reaction(s) of TGase, in this Thesis a number of TGase-mediated reactions have been studied using proteins of known structure and dynamics, as apomyoglobin (apoMb), egg-white lysozyme (LYS) and bovine pancreatic ribonuclease A (RNase).Amino- as well amido-ligands have been used in the TGase-mediated reactions, so that it was possible to analyse the specificity of modification of both Gln and Lys in the examined proteins. We have found an almost strict specificity of TGase-mediated reactions at the level Gln91 of apoMb, a residue embedded in the highly flexible or unfolded helix-F of the holo protein, as given by previous NMR measurements and limited proteolysis data. Also a Gln-mimicking ligand can be covalently linket by TGase at a Lys residue of the same chain region. Thus, we concluded that local enhanced flexibility or even fully local unfolding dictates the site-specific reaction with TGase. While RNase can be selectively modified by using an amido-ligand at the level of the ɛ-amino group of Lys1, a similar Lys1 of LYS was instead fully unreactive. It was possible to relate this finding to the flexibility and rigidity of the N-terminal region of RNase and LYS, respectively, on the basis of the crystallographically determined B-factor values (a measure of chain flexibility) of these two proteins. A nicked species of RNase with the single peptide bond Asn34-Leu35 cleaved (RNase Th1) and a LYS derivative with a single disulfide bridge reduced among the four of native LYS (LYSCM6, 127) were shown to be much more reactive in the TGase-mediated reactions than the parent intact proteins, in agreement with their enhanced flexibility or partial unfolding. Moreover, we could demonstrate that the sites or regions susceptible to TGase reactions are also prone to limited proteolysis phenomena, implying that both TGase and a protease require some local unfolding for a site-specific enzymatic reaction. Indeed, this in keeping with view that the biorecognition phenomenon is similar for both enzymes, considering also the fact that TGase acts as a reverse protease (amide synthesis instead of hydrolysis).An interesting outcome of these studies resides in the fact that we can envisage a novel enzymatic method of covalent coupling of an amino-polymer as poly(ethylene)glycol (PEG) to specific Gln residue(s) of proteins of pharmaceutical interest. Indeed, using TGase and an amino derivative of PEG (PEG-NH2), it was possible to prepare homogeneous PEGylated derivatives of apoMb, human growth hormone (hGH) and granulocyte colony-stimulating factor (G-CSF). Overall, we have interpreted our findings as indicating that the selective TGase-mediated reactions require a flexible or unfolded polypeptide substrate. Therefore, it is possible to predict the sites of TGase attack on a protein substrate, provided that its structure and dynamics are known. Considering the increasing relevance of PEGylated protein drugs and the high regulatory demands for their approval, it can be anticipated that the innovative methods for the site-specific PEGylation of proteins using TGase will be considered a useful advance in the methodologies used for protein modification." @default.
• W51144307 created "2016-06-24" @default.
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• W51144307 date "2011-01-27" @default.
• W51144307 modified "2023-09-27" @default.
• W51144307 title "Site-specific modification of proteins by transglutaminase" @default.
• W51144307 hasPublicationYear "2011" @default.
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