Protein stapling via azide–alkyne ligation

Abdeljabbar, Diya M.; Piscotta, Frank J.; Zhang, Siyan; Link, A. James

doi:10.1039/c4cc06528j

Cited by 11 publications

(10 citation statements)

References 21 publications

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“…Key advantages of this chemistry include its chemoselectivity, spatially controlled reactivity, and efficient and spontaneous formation of the cross-link at the posttranslational level and in the intracellular space. The latter feature allows for the production of the stapled proteins inside cells and eliminates the need for protein manipulation during or after purification (48). Whereas R-GPS has been validated using the O2beY/Cys cross-linking method, we expect this approach can be extended to other noncanonical amino acids and/or other chemistries useful for protein cross-linking (49-52).…”

Section: Discussionmentioning

confidence: 99%

Enzyme stabilization via computationally guided protein stapling

Moore

Zorine

Hansen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 99%

Enzyme stabilization via computationally guided protein stapling

Moore

Zorine

Hansen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…There is also the potential to control the catalytic function of enzymes with introduced disulfide bonds through their redox states, 138 or even potentially through microwave irradiation. 141 Other types of crosslinking technologies are also being developed, [142][143][144] including those that can be genetically encoded. 145,146 Some of the most interesting cross-linkers are those sensitive to environmental conditions.…”

Section: Controlling Enzyme Structural Dynamics Through Covalent Consmentioning

confidence: 99%

Engineered control of enzyme structural dynamics and function

2018

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Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.

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“…[29] Azides also have the advantage of being ac onduitt oo ther functionalities through bioorthogonal click cycloaddition reactionsw ith alkynes. [30,31] In fact, it is the bioconjugation applications [32][33][34][35][36] of azido-UAA that are most commonly employed, whereas their abilityt oa lso act as an IR reporterh as been underutilized, with some notable exceptions. [20,23,[37][38][39][40] Azido-UAA might be more widely employed as both IR reporters and bioconjugationconduitsifan efficient and scalable synthesis was developed from readily available andr elatively inexpensive starting materials.…”

Section: Introductionmentioning

confidence: 99%

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

Tookmanian

Phillips-Piro

Fenlon

et al. 2015

Chemistry A European J

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An unnatural amino acid, 4-(2-azidoethoxy)-l-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.

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Protein stapling via azide–alkyne ligation

Cited by 11 publications

References 21 publications

Enzyme stabilization via computationally guided protein stapling

Enzyme stabilization via computationally guided protein stapling

Engineered control of enzyme structural dynamics and function

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

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