Clickable glutathione using tetrazine-alkene bioorthogonal chemistry for detecting protein glutathionylation.
Protein glutathionylation is one of the major cysteine oxidative modifications in response to reactive oxygen species (ROS). We recently developed a clickable glutathione approach for detecting glutathionylation by using a glutathione synthetase mutant (GS M4) that synthesizes azido-glutathione (γGlu-Cys-azido-Ala) in situ in cells. In order to demonstrate the versatility of clickable glutathione and to increase the chemical tools for detecting glutathionylation, we sought to develop clickable glutathione that uses tetrazine-alkene bioorthogonal chemistry. Here we report two alkene-containing glycine surrogates (allyl-Gly and allyl-Ser) for the biosynthesis of clickable glutathione and their use for detection, enrichment, and identification of glutathionylated proteins. Our results provide chemical tools (allyl-Gly and allyl-Ser for GS M4) for versatile characterization of protein glutathionylation. In addition, we show that the active site of GS can be tuned to introduce a small size chemical tag on glutathione for exploring glutathione function in cells.
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
Dilini N
DN
Kekulandara
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
Kusal T G
KT
Samarasinghe
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
Dhanushka N P
DN
Munkanatta Godage
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. yahn@chem.wayne.edu.
Young-Hoon
YH
Ahn
D000475:Alkenes / Q000737:chemistry*; D020134:Catalytic Domain; D057930:Click Chemistry; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism*; D005981:Glutathione Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*
D000475:Alkenes; D011506:Proteins; D005981:Glutathione Synthase; D005978:Glutathione; D003545:Cysteine