Can misfolded enzymes be beneficial? Yes, they can

M. Marek1,2, A. Kunka1,2, S. M. Marques1,2, M. Havlasek 1,2, M. Vasina 1,2, J. Planas-Igleasias1,2, D. Bednar1,2, Z. Prokop1,2, J. Damborsky1,2

1Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

2International Clinical Research Center, St. Annes University Hospital Brno, Pekarska 53, 65691 Brno, Czech Republic

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 = 73.5C; ΔTm > 23C) 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.


1. Markova K., Chmelova K., Marques S. M., Carpentier P., Bednar D., Damborsky J., Marek M. (2020). Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst. Chemical Science 11, (2020), 11162-11178.

2. Markova K., Kunka, A., Chmelova K., Havlasek, M., Babkova, P., Marques, S. M., Vasina M., Planas-Iglesias J., Chaloupkova, R., Bednar D., Prokop, Z., Damborsky J., Marek M. (2021). Computational enzyme stabilization can affect folding energy landscapes and lead to catalytically enhanced domain-swapped dimers. ACS Catalysis, 11, (2021), 12864-12885.


This work was supported by the Czech Science Foundation (22-09853S).