Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck

Napoli F, Guan J-Y, Arnaud C-A, Macek P, Fraga H, Breyton C, Schanda P. 2024. Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck. Magnetic Resonance. 5(1), 33–49.

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Journal Article | Published | English

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Author
Napoli, FedericoISTA ; Guan, Jia-Ying; Arnaud, Charles-Adrien; Macek, Pavel; Fraga, Hugo; Breyton, Cécile; Schanda, PaulISTA
Department
Abstract
Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.
Publishing Year
Date Published
2024-04-19
Journal Title
Magnetic Resonance
Acknowledgement
We thank Dominique Madern (IBS Grenoble) for providing the plasmid for MalDH and feedback on the article, Alicia Vallet for excellent support at the Grenoble NMR facility, and Petra Rovo and Margarita Valhondo at the IST Austria NMR Service Unit. We thank Dorothea Anrather in the mass spectrometry facility of Max Perutz Labs for the mass spectrometry analysis using the instruments of the Vienna BioCenter Core Facilities (VBCF). We are grateful to Jean-Pierre Andrieu (Plateforme Seq3A, IBS Grenoble) for the analysis of the amino acid composition of the in-house-prepared lysates. We are grateful to Rasmus Linser (Technical University Dortmund) for sharing a paper draft describing a similar study. This work was supported by the Austrian Science Fund (FWF; project number I5812-B). We thank Tobias Schubeis (Lyon) and the reviewers for constructive input. This research has been supported by the Austrian Science Fund (grant no. I5812-B). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for 40 Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, 45 CEA). Charles-Adrien Arnaud was funded by GRAL.
Volume
5
Issue
1
Page
33-49
ISSN
IST-REx-ID

Cite this

Napoli F, Guan J-Y, Arnaud C-A, et al. Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck. Magnetic Resonance. 2024;5(1):33-49. doi:10.5194/mr-5-33-2024
Napoli, F., Guan, J.-Y., Arnaud, C.-A., Macek, P., Fraga, H., Breyton, C., & Schanda, P. (2024). Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck. Magnetic Resonance. Copernicus Publications. https://doi.org/10.5194/mr-5-33-2024
Napoli, Federico, Jia-Ying Guan, Charles-Adrien Arnaud, Pavel Macek, Hugo Fraga, Cécile Breyton, and Paul Schanda. “Deuteration of Proteins Boosted by Cell Lysates: High-Resolution Amide and Ha Magic-Angle-Spinning (MAS) NMR without the Reprotonation Bottleneck.” Magnetic Resonance. Copernicus Publications, 2024. https://doi.org/10.5194/mr-5-33-2024.
F. Napoli et al., “Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck,” Magnetic Resonance, vol. 5, no. 1. Copernicus Publications, pp. 33–49, 2024.
Napoli F, Guan J-Y, Arnaud C-A, Macek P, Fraga H, Breyton C, Schanda P. 2024. Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck. Magnetic Resonance. 5(1), 33–49.
Napoli, Federico, et al. “Deuteration of Proteins Boosted by Cell Lysates: High-Resolution Amide and Ha Magic-Angle-Spinning (MAS) NMR without the Reprotonation Bottleneck.” Magnetic Resonance, vol. 5, no. 1, Copernicus Publications, 2024, pp. 33–49, doi:10.5194/mr-5-33-2024.
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2024-05-22
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