Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms

Journal of the American Chemical Society
Daniel R DempseyPhilip A Cole

Abstract

N-Hydroxysuccinimide (NHS)-esters are widely used to label proteins nonselectively 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 substrates, 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.

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Citations

Oct 4, 2019·The Journal of Biological Chemistry·Hanjie JiangPhilip A Cole
Jan 1, 2021·Journal of the American Society for Mass Spectrometry·Yang TianHui Ye
Mar 23, 2019·Current Opinion in Structural Biology·Philip A ColeHwan Bae
Jul 3, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Lucia De RosaLuca Domenico D'Andrea
Oct 30, 2019·Chemical Reviews·Sonisilpa MohapatraTaekjip Ha
Oct 10, 2021·Nature Structural & Molecular Biology·Daniel R DempseyPhilip A Cole

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