BPS2026 – From ubiquitin to barrel: Computational design of a de novo protein fold-switch

Protein fold-switches play key roles in diverse biological processes and display striking structural diversity, making them attractive targets for designing programmable proteins with new functions. However, their de novo design remains a formidable challenge. To investigate the determinants of fold switching, we combined sequence-, structure-, and dynamics-based analyses of known switches using physics-based molecular-dynamics simulations and free-energy calculations (GROMACS/pmx) and advanced machine-learning tools (AF3, Foldseek). This integrated strategy enabled the identification of residues and interaction networks critical for switching and allowed us to introduce additional layers of control into existing switches. Guided by these insights, we designed a new ubiquitin-based fold switch, UBI-Switch (Ubiquitin-Barrel Switch I), which reversibly transitions between the canonical ubiquitin fold and a barrel-like conformation. This engineered switch offers a potential means to modulate non-covalent interactions of ubiquitin-binding proteins and provides a framework for creating tunable fold-switching proteins.