| Autor: |
Ablat G; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Lawton N; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Alam R; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Haynes BA; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Hossain S; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Hicks T; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Evans SL; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Jarvis JA; Randall Centre for Cell and Molecular Biophysics and Centre for Biomolecular Spectroscopy, King's College London, New Hunts House, London SE1 1UL, U.K., Nott TJ; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Isaacson RL; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K., Müller MM; Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, U.K. |
| Abstrakt: |
Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited, as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired l-configuration and a roughly equal amount of the undesired d-isomer. Here, we describe a simple remedy for this issue: removal of the d-isomer via proteolysis using a d-stereoselective peptidase, alkaline d-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the d-isomer of epimeric residues within histone H3, GFP, Ddx4, and SGTA, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications. |
