Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 237, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2023.124141
Keywords
Protein thermal stability; Protein engineering; Melting temperature; Glutamine-binding protein; Hydroxyacyl-coenzyme A dehydrogenase; Tyrosine-protein kinase SYK
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Optimizing the thermal stability of multidomain proteins is crucial for the development of industrial enzymes and biologics. This study demonstrates that domain-based engineering followed by domain recombination can significantly increase the stability of multidomain proteins. Independent enhancement of the thermostability of constituent domains improves the overall stability of the protein, as explained by crystal structure and AlphaFold-predicted models. This research suggests that domain-based modular engineering offers valuable guidance for multidomain protein engineering by reducing sequence space and experimental errors.
Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and biologics. Herein, we demonstrate that multidomain proteins can be stabilized significantly using domain-based engineering followed by the recombination of the optimized domains. Domain-level analysis of designed protein variants with similar structures but different thermal profiles showed that the independent enhancement of the thermostability of a constituent domain improves the overall stability of the whole multidomain protein. The crystal structure and AlphaFold-predicted model of the designed proteins via domain-recombination provided a molecular explanation for domain-based stepwise stabilization. Our study suggests that domain-based modular engineering can minimize the sequence space for calculations in computational design and experimental errors, thereby offering useful guidance for multidomain protein engineering.
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