In Cellulo Cysteine Umpolung for Protein Structure Probing

Journal of the American Chemical Society, vol. 147, pp. 35587–35594

Abstract

High Resolution Image Download MS PowerPoint Slide Proteome-wide intramolecular cross-linking of amino acids allows for protein structure probing and is often achieved by reactive intermediates generated in situ . For example, carbene insertion chemistry forms versatile products but relies on two-step protocols that first introduce a photoreactive group and then generate a reactive intermediate via UV light irradiation. Alternatively, direct cross-linking with bifunctional electrophilic reagents enables modification of natural amino acids in a single transformation but proceeds via intermediates with a sufficiently long half-life in solution to capture unfavorable protein conformations. Herein, we report the use of commercially available vinyl thianthrenium tetrafluoroborate ( VTT ) for a single-step cross-linking strategy based on the umpolung of native cysteinyl thiols to electrophilic episulfonium ions in cells. We demonstrate that the umpolung reaction proceeds within minutes in various cell types. Even when competing with cysteine-reactive maleimide or iodoacetamide reagents, the fast cellular uptake of VTT enables efficient intracellular labeling and ensures the formation of products with a short and stable ethylene linker between cysteine and nucleophilic natural amino acids. We anticipate that the fast generation of episulfonium ions, which enable cross-linking without exogenous activation, will assist researchers in predicting protein structures based on quantitative proteome-wide information generated in cellulo .

Authors 8

1. Philipp Hartmann Aachen Institute of Organic Chemistry

   Max-Planck-Institut für Kohlenforschung · RWTH Aachen University

   Affiliation as printed

   Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany

   Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

2. Kostiantyn Bohdan Aachen Institute of Organic Chemistry

   Max-Planck-Institut für Kohlenforschung · RWTH Aachen University

   Affiliation as printed

   Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany

   Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

3. Lara Vogelsang

   Bielefeld University

   Affiliation as printed

   Biochemistry and Physiology of Plants, Faculty of Biology, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany

4. Dario Marchionni

   Max-Planck-Institut für Kohlenforschung

   Affiliation as printed

   Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

5. Christian Preisinger Aachen

   RWTH Aachen University

   Affiliation as printed

   Proteomics Facility, Interdisciplinary Centre for Clinical Research (IZKF)

   RWTH Aachen University

6. Alessandro Vetere

   Max-Planck-Institut für Kohlenforschung

   Affiliation as printed

   Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

7. Karl‐Josef Dietz

   Bielefeld University

   Affiliation as printed

   Biochemistry and Physiology of Plants, Faculty of Biology, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany

8. Tobias Ritter corresponding

   Max-Planck-Institut für Kohlenforschung

   Affiliation as printed

   Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany