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Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α

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Title: Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α
Authors: Rania Ghouil, Chafiaa Bouguechtouli, Hélène Chérot, Agathe Marcelot, Maxime Roche, Francois-Xavier Theillet
Source: Journal of Magnetic Resonance Open, Vol 16, Iss , Pp 100126- (2023)
Publisher Information: Elsevier, 2023.
Publication Year: 2023
Collection: LCC:Medical physics. Medical radiology. Nuclear medicine
LCC:Physics
Subject Terms: Kinase, p38, Cell-free expression, Deuteration, In-cell NMR, Medical physics. Medical radiology. Nuclear medicine, R895-920, Physics, QC1-999
More Details: Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10.
Document Type: article
File Description: electronic resource
Language: English
ISSN: 2666-4410
Relation: http://www.sciencedirect.com/science/article/pii/S2666441023000341; https://doaj.org/toc/2666-4410
DOI: 10.1016/j.jmro.2023.100126
Access URL: https://doaj.org/article/3038acd041dd4e6297a82050afa43182
Accession Number: edsdoj.3038acd041dd4e6297a82050afa43182
Database: Directory of Open Access Journals
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  Data: Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α
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  Data: Journal of Magnetic Resonance Open, Vol 16, Iss , Pp 100126- (2023)
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  Data: Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10.
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      – SubjectFull: Cell-free expression
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      – TitleFull: Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α
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