Enzyme stabilization via computationally guided protein stapling

Abstract

Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (Δ = +18.0 °C and Δ = +16.0 °C) as well as increased stability against chemical denaturation [Δ (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.

Enhancement of protein stability by an additional disulfide bond designed in human neuroglobin

“…43 Fasan and co-workers showed that construction of an articial thioether bond between a non-natural amino acid of O-2-bromoethyl Tyr and Cys in Mb may increase $0.24 M for C m upon Gdn$HCl-induced unfolding. 57 Moreover, Hollenberg and co-workers constructed a de novo disulde bond close to the heme center in Cyt P450 2B1 by a double mutation of Y309C/ S360C, which reduced the protein plasticity and ne-tuned the metabolic activity. 58 These observations suggest that the location for engineering a covalent bond is crucial for the enhancement of stability, as well as functional regulation.…”

Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin

“…In the future, further advantage might be gained by engineering redox-stable disulfide analogs, such as has been explored in the case of sunflower trypsin inhibitor, a very small canonical inhibitor accessible through total synthesis (42,43). This type of engineering would require incorporation of stable cross-links into proteins through the use of genetically encoded, noncanonical amino acids (26,44). We predict that such disulfide-engineered proteins with enhanced stability could lead to the development of a new generation of biopharmaceuticals.…”

Molecular Dynamics Simulation and Kinetic Study of Fluoride Binding to V21C/V66C Myoglobin with a Cytoglobin-like Disulfide Bond

“…This is especially the case for heme protein design, which has received much attention in the last few decades, and various approaches have been established for rational design, such as the introduction of non-heme metal ions and unnatural amino acids, and the use of heme mimics to act as an active site [1][2][3][4][5][6][7][8][9][10][11][12]. Importantly, computer modeling and molecular dynamics (MD) simulation play key roles in guiding the protein design [13][14][15][16][17][18]. For example, computer simulation was successfully applied to design a non-heme iron binding site in the heme pocket of myoglobin (Mb), which converted an O 2 carrier into a functional nitric oxide reductase [19].…”