Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity.

TitleEngineering the kinetic stability of a β-trefoil protein by tuning its topological complexity.
Publication TypeJournal Article
Year of Publication2023
AuthorsAnderson DM, Jayanthi LP, Gosavi S, Meiering EM
JournalFront Mol Biosci
Date Published2023

Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two β-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.

Alternate JournalFront Mol Biosci
PubMed ID36845544
PubMed Central IDPMC9945329
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