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Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis
Guljannat Ablat1, Neev Lawton1, Ruqaiya Alam1
1Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K.
Journal of the American Chemical Society
|July 31, 2024
Summary
Chemical mutagenesis using dehydroalanine (Dha) creates epimeric mixtures. A new method uses alkaline d-peptidase (AD-P) to remove the undesired d-isomer, enabling stereocontrolled protein engineering.
Area of Science:
- Protein Engineering
- Chemical Biology
- Biochemistry
Background:
- Chemical mutagenesis via dehydroalanine (Dha) allows site-specific protein modification.
- Current Dha methods yield epimeric mixtures (l- and d-isomers), limiting applications.
- Stereochemical control is crucial for precise protein engineering.
Purpose of the Study:
- To develop a method for stereoselective removal of undesired d-isomers in Dha-modified proteins.
- To enable precise installation of non-natural amino acids with controlled stereochemistry.
Main Methods:
- Utilized alkaline d-peptidase (AD-P), a d-stereoselective peptidase.
- Applied AD-P for proteolysis of d-epimers in histone H3, GFP, Ddx4, and SGTA.
- Demonstrated selective cleavage of d-isomers within target proteins.
Main Results:
- AD-P effectively and selectively removed the d-isomer of epimeric residues.
- Successful stereochemical control was achieved in modified proteins.
- The method is compatible with various protein targets.
Conclusions:
- Stereoselective chemoenzymatic mutagenesis using AD-P offers a simple solution to epimeric mixtures.
- This approach facilitates the installation of non-natural amino acids with high stereochemical fidelity.
- The method holds significant potential for advancing protein engineering and chemical biology.
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