The functionality of a protein catalyst (enzyme) depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications [1].
Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase fold enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, making extensive non-covalent interactions between the intertwined misfolded protomers [2]. The existence of domain-swapped dimers in a solution is confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers could be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo [2]. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation protein catalysts.
This work was supported by the Czech Science Foundation (22-09853S).