title: A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions.
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.
authors.affiliation: Chemistry Department, University of Michigan, 930 N. University Avenue, Ann Arbor, MI, 48109, USA.|Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI, 48109, USA.
authors.forename: Cassandra M
authors.initials: CM
authors.lastname: Joiner
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI, 48109, USA.
authors.forename: Meghan E
authors.initials: ME
authors.lastname: Breen
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI, 48109, USA.
authors.forename: James
authors.initials: J
authors.lastname: Clayton
authors.affiliation: Chemistry Department, University of Michigan, 930 N. University Avenue, Ann Arbor, MI, 48109, USA.|Program in Chemical Biology, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI< postCode/>48109, USA.|Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI, 48109, USA.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Mapp
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001386:Azides / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000480:Alkynes; D000596:Amino Acids; D001386:Azides; D001577:Benzophenones; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D001710:Biotin; C047723:benzophenone; D003300:Copper
keywords: bioorthogonal labeling; click chemistry; photo-crosslinking; protein-protein interactions; unnatural amino acids