Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes

Mathew Stanley, Cong Han, Axel Knebel, Paul Murphy, Natalia Shpiro, Satpal Virdee (Lead / Corresponding author)

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    15 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)1542-1554
    Number of pages13
    JournalACS Chemical Biology
    Volume10
    Issue number6
    Early online date6 Apr 2015
    DOIs
    Publication statusPublished - 19 Jun 2015

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