Improving split reporters of protein-protein interactions through orthology-based protein engineering

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Protein-protein interactions (PPI) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of RspA-splitFAST, an improved reporter displaying higher brightness, lower self-complementation and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, RspA-splitFAST is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

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  1. Looks like your PYP ortholog detection started with a sequence identity-based search. Have you also done a protein structure based search, eg via foldseek, to identify potential orthologs that may have low sequence similarly but high structural conservation to uncover additional candidates with beneficial properties?