Skip to Main content Skip to Navigation
Journal articles

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

Abstract : Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.
Complete list of metadatas

https://hal-pasteur.archives-ouvertes.fr/pasteur-02883959
Contributor : Nicolas Wolff <>
Submitted on : Monday, June 29, 2020 - 3:35:08 PM
Last modification on : Saturday, October 3, 2020 - 3:12:05 AM

Links full text

Identifiers

Citation

Renaud Vincentelli, Katja Luck, Juline Poirson, Jolanta Polanowska, Julie Abdat, et al.. Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. Nature Methods, Nature Publishing Group, 2015, 12 (8), pp.787-793. ⟨10.1038/nmeth.3438⟩. ⟨pasteur-02883959⟩

Share

Metrics

Record views

42