Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives

被引:18
|
作者
Larda, Sacha Thierry [1 ]
Pichugin, Dmitry [1 ]
Prosser, Robert Scott [1 ,2 ]
机构
[1] Univ Toronto, Dept Chem, UTM, Mississauga, ON L5L 1C6, Canada
[2] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada
关键词
NUCLEAR-MAGNETIC-RESONANCE; PK(A) VALUES; TYROSINE BIOCONJUGATION; REDUCTIVE ALKYLATION; SURFACE-AREAS; CROSS-LINKING; NMR; CHEMISTRY; RATIONALIZATION; CONFORMATIONS;
D O I
10.1021/acs.bioconjchem.5b00457
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 a-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain N epsilon- 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. F-19 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. F-19 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.
引用
收藏
页码:2376 / 2383
页数:8
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