2026/01/01 by Antoine Guyot, Emma Maillard, Kelly Ferreira-Pinto +8 · 1 voice
Biochemistry, Genetics and Molecular Biology · #Biochemical and Structural Characterization #Bioinformatics and Genomic Networks #Machine Learning in Bioinformatics #Protein Structure and Dynamics #RNA and protein synthesis mechanisms #vaccines and immunoinformatics approaches
paper · doi:10.34133/csbj.0098
openalex publication_date 2026/01/01 · openalex created_date 2026/04/20 · openalex updated_date 2026/07/04
ABSTRACT Characterizing mutation effects on protein-protein interactions (PPIs) is crucial for elucidating protein structure and function. Massively parallel PPI variant analyses such as deep mutational scanning (DMS) enable interface identification and generate datasets for machine learning. In cellulo strategies such as two-hybrid systems provide straightforward access to such data, but reliability depends on quantitative properties. Here, we show that existing bacterial two-hybrid (B2H) systems have limitations constraining accurate dataset generation. We engineered and benchmarked optimized quantitative B2H (qB2H) alternatives, enabling strain-independent assays, improved metrics, and generation of high-quality datasets. We demonstrate qB2H utility through interface mapping and binder optimization. Perturbation analysis of single-site variants accurately recovered known ASF1 complex contact positions, matching crystallographic data. Integration of generative AI–based design yielded an ASF1-binding peptide with a 70-fold increase in affinity. qB2H offers to R&D scientists a robust, reusable platform for quantitative PPI analysis, enabling both rational protein engineering and data-driven discovery.