title: A genetically encoded acrylamide functionality.
abstract: Nε-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNACUAPyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Nε-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Nε-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
authors.forename: Yan-Jiun
authors.initials: YJ
authors.lastname: Lee
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Raymond
authors.forename: Yu
authors.initials: Y
authors.lastname: Zeng
authors.forename: Xinqiang
authors.initials: X
authors.lastname: Fang
authors.forename: Karen L
authors.initials: KL
authors.lastname: Wooley
authors.forename: Wenshe R
authors.initials: WR
authors.lastname: Liu
mesh_terms: D020106:Acrylamide / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008623:Mercaptoethanol / Q000737:chemistry*; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D020106:Acrylamide; D008623:Mercaptoethanol; D000604:Amino Acyl-tRNA Synthetases; D005978:Glutathione; D008239:Lysine