High-throughput characterization of protein-protein interactions by reprogramming yeast mating

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• High-throughput screening methods are crucial for characterizing protein-protein interactions and interaction networks.
• Existing methods lack the ability for quantitative, library-on-library characterization in a modifiable extracellular environment.

Purpose of the Study:

• To develop a novel method, synthetic agglutination (SynAg), for high-throughput, quantitative screening of protein-protein interactions.
• To enable the characterization of protein interaction networks within a defined and modifiable extracellular environment.

Main Methods:

• Reprogramming sexual agglutination in *Saccharomyces cerevisiae* to link mating efficiency with protein interaction strength.
• Utilizing induced chromosomal translocation to pair binding protein barcodes for single-pot screening of thousands of interactions.
• Introducing soluble peptides to selectively disrupt specific interactions within a network.

Main Results:

• SynAg demonstrates a log-linear relationship between mating efficiency and protein binding strength (K_ds from pM to μM) for 89 characterized interactions.
• Thousands of distinct protein-protein interactions can be screened simultaneously using SynAg.
• Selective disruption of interactions by up to 800-fold was achieved using soluble peptides, demonstrating environmental modifiability.

Conclusions:

• SynAg provides a powerful platform for the high-throughput, quantitative characterization of protein-protein interactions.
• The method allows for the study of interaction networks in a defined and modifiable extracellular milieu.
• SynAg is scalable for library-on-library interaction analysis.