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. 2017 Nov 21;114(47):12472-12477.
doi: 10.1073/pnas.1708907114. Epub 2017 Nov 6.

Enzyme Stabilization via Computationally Guided Protein Stapling

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Enzyme Stabilization via Computationally Guided Protein Stapling

Eric J Moore et al. Proc Natl Acad Sci U S A. .
Free PMC article


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 (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chemical denaturation [ΔCm (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.

Keywords: Rosetta macromolecular modeling; computational protein design; myoglobin; noncanonical amino acids; protein thermostabilization.

Conflict of interest statement

The authors declare no conflict of interest.


Fig. 1.
Fig. 1.
Computational design approach. (A) Stapling reaction between the noncanonical amino acid O-2-bromoethyl tyrosine and cysteine resulting in a chemically stable thioether bond. (B) Structure of Mb (Protein Data Bank ID code 1JP9) highlighting helices A–H (Left). The active-site heme group and metal-coordinating histidine residue are shown as sticks. Conformational ensemble of the modeled thioether linkage used to find compatible locations for stapling (Right). (C) Models showing locations of covalent staples between A–H and B–G helices in designs sMb1 through sMb9.
Fig. 2.
Fig. 2.
Characterization of stapled Mb(H64V,V68A) variants (sMb variants). (A and B) SDS/PAGE gel (A) and MALDI-TOF MS spectrum (B) of Mb(H64V,V68A) and representative sMb variants. c, cross-linked; c(2×), doubly cross-linked; nc, not cross-linked. Calculated masses: Mb(H64V,V68A): 18,474 Da; sMb2 (c): 18,500 Da; sMb4 (c): 18,472 Da; sMb5 (c): 18,594 Da; sMb10 [c(2×)]: 18,621 Da; sMb13 [c(2×)]: 18,612 Da. (C) Thermal denaturation curves for Mb(H64V,V68A) and selected stapled variants as measured via CD at 220 nm (Tm determination). (D) Heat-induced inactivation curves (heme loss) for the same proteins as determined by decrease of Soret band signal (408 nm) after incubation (10 min) at variable temperatures (T50 determination). See SI Appendix, Figs. S5–S7 for additional data. (E and F) Catalytic activity (E) and enantioselectivity (F) of Mb(H64V,V68A) and stapled variants in styrene cyclopropanation reactions with EDA in buffer only and in the presence of 30% vol/vol ethanol. Relative activities refer to normalized catalytic turnovers (TON) relative to TON measured with Mb(H64V,V68A) in buffer only reactions. See SI Appendix, Figs. S10–S12 for related data with other organic cosolvents. Rel Int, relative intensity.

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