title: Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes.
abstract: Transthiolation is a fundamental biological reaction and is utilized by many enzymes involved in the conjugation of ubiquitin and ubiquitin-like proteins. However, tools that enable selective profiling of this activity are lacking. Transthiolation requires cysteine-cysteine juxtaposition; therefore a method that enables irreversible "stapling" of proximal thiols would facilitate the development of novel probes that could be used to profile this activity. Herein, we characterize biocompatible chemistry that enables sequential functionalization of cysteines within proteins at a single atomic center. We use our method to develop a new class of activity-based probe that profiles transthiolation activity of human E1 activating enzymes. We demonstrate use in vitro and in situ and compatibility with competitive activity-based protein profiling. We also use the probe to gain insight into the determinants of transthiolation between E2 and a RING-in-between-RING (RBR) E3 ligase. Furthermore, we anticipate that this method of thiol functionalization will have broad utility by enabling simple redox-stable cross-linking of proximal cysteines in general.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Mathew
authors.initials: M
authors.lastname: Stanley
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Cong
authors.initials: C
authors.lastname: Han
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Murphy
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Natalia
authors.initials: N
authors.lastname: Shpiro
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004123:Dimethylamines / Q000737:chemistry; D005030:Ethylenes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004123:Dimethylamines; D005030:Ethylenes; D013438:Sulfhydryl Compounds; C471538:UBA1 protein, human; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D044764:Ubiquitin-Activating Enzymes; D003545:Cysteine; C548965:tetrakis(dimethylamino)ethylene