Structural and energetic analysis of stabilizing indel mutations

Amino acid insertions and deletions (indels) are among the most common protein mutations and necessitate changes to a protein's backbone geometry. Examining how indels affect protein folding stability (and especially how indels can increase stability) can help reveal the role of backbone energetics on stability and introduce new protein engineering strategies. Tsuboyama et al. measured folding stability for 57,698 single amino acid insertion or deletion mutants in 405 small domains, and this analysis identified 103 stabilizing mutants (ΔΔG_unfolding > 1 kcal/mol). Here, we use computational modeling to analyze structural and energetic changes for these stabilizing indel mutants. We find that stabilizing indel mutations tend to have local structural effects and that stabilizing deletions (but less so insertions) are often found in regions of high backbone strain. We also find that stabilizing indels are typically correctly classified as stabilizing by the Rosetta energy function (which explicitly models backbone energetics), but not by an inverse folding (ESM-IF)-based analysis (Cagiada et al. 2024) which predicts absolute stability (ΔG_unfolding).