title: N-terminal protein modification by substrate-activated reverse proteolysis.
abstract: Although site-specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio- and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site-specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N-terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen-like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.
authors: Institute of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle/Saale (Germany).
authors: Sandra
authors: S
authors: Liebscher
authors: Michael
authors: M
authors: Schöpfel
authors: Tobias
authors: T
authors: Aumüller
authors: Ariunkhur
authors: A
authors: Sharkhuukhen
authors: Andreas
authors: A
authors: Pech
authors: Eva
authors: E
authors: Höss
authors: Christoph
authors: C
authors: Parthier
authors: Günther
authors: G
authors: Jahreis
authors: Milton T
authors: MT
authors: Stubbs
authors: Frank
authors: F
authors: Bordusa
mesh_terms: D055162:Biocatalysis; D021983:Cyclophilins / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D011506:Proteins; C081786:cyclophilin B; D014357:Trypsin; D021983:Cyclophilins
keywords: biocatalysis; bioconjugation; protein modification; proteinases; reverse proteolysis