1. Academic Validation
  2. A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions

A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions

  • Chembiochem. 2017 Jan 17;18(2):181-184. doi: 10.1002/cbic.201600578.
Cassandra M Joiner 1 2 Meghan E Breen 2 James Clayton 2 Anna K Mapp 1 3 2
Affiliations

Affiliations

  • 1 Chemistry Department, University of Michigan, 930 N. University Avenue, Ann Arbor, MI, 48109, USA.
  • 2 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI, 48109, USA.
  • 3 Program in Chemical Biology, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI< postCode/>48109, USA.
Abstract

In vivo covalent chemical capture by using photoactivatable unnatural Amino acids (UAAs) is a powerful tool for the identification of transient protein-protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4-Gal80 transcriptional complex. Post-crosslinking, the Gal4-Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin-azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole-cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.

Keywords

bioorthogonal labeling; click chemistry; photo-crosslinking; protein-protein interactions; unnatural amino acids.

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