37 research outputs found
Site-Specific Protein Labeling with <i>N</i>‑Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms
<i>N</i>-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