Systematic characterization of indel variants using a yeast-based protein folding sensor

Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.