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High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale

Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Y...

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Detalles Bibliográficos
Autores principales: Schulte, Clemens, Khayenko, Vladimir, Nordblom, Noah Frieder, Tippel, Franziska, Peck, Violetta, Gupta, Amit Jean, Maric, Hans Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753147/
https://www.ncbi.nlm.nih.gov/pubmed/33364586
http://dx.doi.org/10.1016/j.isci.2020.101898
Descripción
Sumario:Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.