Site-specific Protein Labeling with NHS-Esters and the Analysis of Ubiquitin Ligase Mechanisms

N-hydroxysuccinimide (NHS)-esters are widely used to label proteins non-selectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transfo...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2018-07, Vol.140 (30), p.9374-9378
Main Authors: Dempsey, Daniel R., Jiang, Hanjie, Kalin, Jay H., Chen, Zan, Cole, Philip A.
Format: Article
Language:English
Online Access:Get full text
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Summary:N-hydroxysuccinimide (NHS)-esters are widely used to label proteins non-selectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transform NHS-esters into site-specific protein labeling on N-terminal Cys residues. MESNA addition converts NHS-esters to chemoselective thioesters for N-Cys modification. This labeling strategy was applied to clarify mechanistic features of the ubiquitin E3 ligase WWP2 including its interaction with one of its substrate, the tumor suppressor PTEN, as well as its autoubiquitination molecularity. We propose that this convenient protein labeling strategy will allow for an expanded application of NHS-esters in biochemical investigation.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.8b05098