Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers
| Authors | |
|---|---|
| Year of publication | 2021 |
| Type | Article in Periodical |
| Magazine / Source | ACS Catalysis |
| MU Faculty or unit | |
| Citation | |
| Web | https://pubs.acs.org/doi/10.1021/acscatal.1c03343 |
| Doi | http://dx.doi.org/10.1021/acscatal.1c03343 |
| Keywords | protein folding; protein design; alpha/beta-hydrolase; haloalkane dehalogenase; domain swapping; energy landscape; oligonicrization; catalytic efficiency; substrate inhibition |
| Description | The functionality of an 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. Here, we reveal a unique example where the computational stabilization of a monomeric alpha/beta-hydrolase enzyme (T-m = 73.5 degrees C; Delta T-m > 23 degrees C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking 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. 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 biocatalysts. |
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