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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

preprint
submitted on 23.01.2021, 19:43 and posted on 27.01.2021, 05:34 by Klara Markova, Antonin Kunka, Klaudia Chmelova, Martin Havlasek, Petra Babkova, Sérgio M. Marques, Michal Vasina, Joan Planas-Iglesias, Radka Chaloupková, David Bednar, Zbynek Prokop, Jiri Damborsky, Martin Marek

The functionality of a protein 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. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase 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, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

History

Email Address of Submitting Author

jiri@cjemi.muni.cz

Institution

Masaryk University

Country

Czech Republic

ORCID For Submitting Author

0000-0002-7848-8216

Declaration of Conflict of Interest

Authors declare no conflict of interest.

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