Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis

Guljannat Ablat; Neev Lawton; Ruqaiya Alam; Bethany A. Haynes; Sabrina Hossain; Thomas Hicks; Sasha L. Evans; James A. Jarvis; Timothy J. Nott; Rivka L. Isaacson; Manuel M. Müller

doi:10.1021/jacs.4c07103

Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis

Guljannat Ablat, Neev Lawton, Ruqaiya Alam, Bethany A. Haynes, Sabrina Hossain, Thomas Hicks, Sasha L. Evans, James A. Jarvis, Timothy J. Nott, Rivka L. Isaacson, Manuel M. Müller^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	22622-22628
Number of pages	7
Journal	Journal of the American Chemical Society
Volume	146
Issue number	32
DOIs	https://doi.org/10.1021/jacs.4c07103
Publication status	Published - 14 Aug 2024

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title = "Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis",

abstract = "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.",

author = "Guljannat Ablat and Neev Lawton and Ruqaiya Alam and Haynes, {Bethany A.} and Sabrina Hossain and Thomas Hicks and Evans, {Sasha L.} and Jarvis, {James A.} and Nott, {Timothy J.} and Isaacson, {Rivka L.} and M{\"u}ller, {Manuel M.}",

note = "Publisher Copyright: {\textcopyright} 2024 The Authors. Published by American Chemical Society.",

year = "2024",

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doi = "10.1021/jacs.4c07103",

language = "English",

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AU - Ablat, Guljannat

AU - Lawton, Neev

AU - Alam, Ruqaiya

AU - Haynes, Bethany A.

AU - Hossain, Sabrina

AU - Hicks, Thomas

AU - Evans, Sasha L.

AU - Jarvis, James A.

AU - Nott, Timothy J.

AU - Isaacson, Rivka L.

AU - Müller, Manuel M.

PY - 2024/8/14

Y1 - 2024/8/14

N2 - 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.

AB - 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.

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JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 32

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Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis

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