Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α
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,...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-12-01
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| Series: | Journal of Magnetic Resonance Open |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666441023000341 |
| _version_ | 1797394748832481280 |
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| author | Rania Ghouil Chafiaa Bouguechtouli Hélène Chérot Agathe Marcelot Maxime Roche Francois-Xavier Theillet |
| author_facet | Rania Ghouil Chafiaa Bouguechtouli Hélène Chérot Agathe Marcelot Maxime Roche Francois-Xavier Theillet |
| author_sort | Rania Ghouil |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T00:24:04Z |
| format | Article |
| id | doaj.art-3038acd041dd4e6297a82050afa43182 |
| institution | Directory Open Access Journal |
| issn | 2666-4410 |
| language | English |
| last_indexed | 2024-03-09T00:24:04Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Magnetic Resonance Open |
| spelling | doaj.art-3038acd041dd4e6297a82050afa431822023-12-12T04:36:36ZengElsevierJournal of Magnetic Resonance Open2666-44102023-12-0116100126Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38αRania Ghouil0Chafiaa Bouguechtouli1Hélène Chérot2Agathe Marcelot3Maxime Roche4Francois-Xavier Theillet5Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, FranceUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, FranceUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, FranceUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, FranceCortecNet, 91940 Les Ulis, FranceUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France; Corresponding author.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.http://www.sciencedirect.com/science/article/pii/S2666441023000341Kinasep38Cell-free expressionDeuterationIn-cell NMR |
| spellingShingle | Rania Ghouil Chafiaa Bouguechtouli Hélène Chérot Agathe Marcelot Maxime Roche Francois-Xavier Theillet Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α Journal of Magnetic Resonance Open Kinase p38 Cell-free expression Deuteration In-cell NMR |
| title | Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α |
| title_full | Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α |
| title_fullStr | Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α |
| title_full_unstemmed | Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α |
| title_short | Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α |
| title_sort | affordable amino acid α β deuteration and specific labeling for nmr signal enhancement evaluation on the kinase p38α |
| topic | Kinase p38 Cell-free expression Deuteration In-cell NMR |
| url | http://www.sciencedirect.com/science/article/pii/S2666441023000341 |
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