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Quantifying domain-ligand affinities and specificities by high-throughput holdup assay
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Quantifying domain-ligand affinities and specificities by high-throughput holdup assay
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Journal Article
Quantifying domain-ligand affinities and specificities by high-throughput holdup assay
2015
Overview
This paper reports an approach to measure equilibrium binding affinities for interacting proteins in high throughput, allowing the rapid and quantitative profiling of the specificity of interaction motifs.
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.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ 631/553
/ 82/56
/ 82/80
/ Biochemistry, Molecular Biology
/ Biomedical Engineering/Biotechnology
/ DNA-Binding Proteins - chemistry
/ High-Throughput Screening Assays
/ Humans
/ Kinetics
/ Ligands
/ Oncogene Proteins, Viral - chemistry
/ Protein Interaction Mapping - methods
/ Protein-protein interactions
/ Proteins
/ Proteome
