2021 journal article

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions

ACS SYNTHETIC BIOLOGY, 10(3), 505–514.

By: K. Bacon n, A. Blain n, J. Bowen n, M. Burroughs n, N. McArthur  n, S. Menegatti n , B. Rao n

author keywords: yeast two-hybrid; protein-protein interactions; protein engineering; yeast surface display; post-translational modification; cDNA library
MeSH headings : Gene Library; Genes, Reporter; Protein Binding; Protein Domains; Protein Interaction Maps; Saccharomyces cerevisiae / genetics; Saccharomyces cerevisiae / metabolism; Smad3 Protein / chemistry; Smad3 Protein / metabolism; Transcription Factors / chemistry; Transcription Factors / metabolism; Two-Hybrid System Techniques
TL;DR: A quantitative yeast-yeast two-hybrid system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD), and it was shown that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein. (via Semantic Scholar)
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Source: Web Of Science
Added: March 29, 2021

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.