title: Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives.
abstract: Indoles and indole-derivatives can be used to site-specifically label proteins on lysine and N-terminal amino groups under mild, nondenaturing reaction conditions. Hen egg white lysozyme (HEWL) and α-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain Nε- and N-terminal amino imines, formed in situ in the presence of formaldehyde. The reaction is highly site-selective, easily controlled by temperature, and does not eliminate the native charge of the protein, unlike many other common lysine-specific labeling strategies. (19)F NMR was used to monitor reaction progression, and in the case of HEWL, unique resonances for each labeled side chain could be resolved. We demonstrate that the indole tags are highly selective for primary amino groups. (19)F NMR demonstrates that each lysine exhibits a different rate of conjugation to indoles making it possible to employ these tags as a means of probing surface topology by NMR or mass spectrometry. Given the site-specificity of this tagging method, the mildness of the reaction conditions (aqueous, buffered, or unbuffered) and the low stoichiometry required for the reaction, indole-derivatives should serve as a valuable addition to the bioconjugation toolkit. We propose that labeling lysine side chains and N-terminal amino groups with indoles is a versatile and general strategy for bioconjugations with substituted indoles having broad implications for protein functionalization.
authors.affiliation: Department of Chemistry, University of Toronto, UTM , 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.
authors.forename: Sacha Thierry
authors.initials: ST
authors.lastname: Larda
authors.affiliation: Department of Chemistry, University of Toronto, UTM , 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Pichugin
authors.affiliation: Department of Chemistry, University of Toronto, UTM , 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.|Department of Biochemistry, University of Toronto , 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Robert Scott
authors.initials: RS
authors.lastname: Prosser
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D054879:Halogenation; D007202:Indicators and Reagents; D007211:Indoles / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry*; D008239:Lysine / Q000032:analysis*; D013058:Mass Spectrometry; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D013194:Staining and Labeling
chemical_list: D007202:Indicators and Reagents; D007211:Indoles; D007768:Lactalbumin; C421864:hen egg lysozyme; D009113:Muramidase; D008239:Lysine