<p>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 (<i>T</i><sub>m</sub> = 73.5°C;
Δ<i>T</i><sub>m</sub> > 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 <i>in vivo</i>. 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.</p>
Correlation between rate of folding, energy landscape, and topology in the folding of a model protein HP-36