title: SUMOylated RanGAP1 prepared by click chemistry.
abstract: Ubiquitin and ubiquitin-like proteins such as SUMO represent important and abundant post-translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189-amino acid fragment of human RanGAP1 (amino acids 398-587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)-catalyzed alkyne-azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole-linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin-like proteins and underline the importance of having alternative approaches to do so.
authors: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149, Münster, Germany.
authors: Nadine D
authors: ND
authors: van Treel
authors: Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149, Münster, Germany.
authors: Henning D
authors: HD
authors: Mootz
mesh_terms: D002384:Catalysis; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D008958:Models, Molecular; D011499:Protein Processing, Post-Translational; D058207:Sumoylation
chemical_list: D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D003300:Copper
keywords: CuAAC; SUMO; bioorthogonal; click chemistry; post‐translational modification; protein chemistry; ubiquitin