Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems

Abhinav Dubey; Nikolay Stoyanov; Thibault Viennet; Sandeep Chhabra; Shantha Elter; Jan Borggräfe; Aldino Viegas; Radosław P. Nowak; Nikola Burdzhiev; Ognyan Petrov; Eric S. Fischer; Manuel Etzkorn; Vladimir Gelev; Haribabu Arthanari

doi:10.1002/anie.202016070

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems

Abhinav Dubey, Nikolay Stoyanov, Thibault Viennet, Sandeep Chhabra, Shantha Elter, Jan Borggräfe, Aldino Viegas, Radosław P. Nowak, Nikola Burdzhiev, Ognyan Petrov, Eric S. Fischer, Manuel Etzkorn^*, Vladimir Gelev^*, Haribabu Arthanari^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

10 Citations (Scopus)

Abstract

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective ¹³C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.

Original language	English
Journal	Angewandte Chemie - International Edition
Volume	60
Issue	25
Pages (from-to)	13783-13787
Number of pages	5
ISSN	1433-7851
DOIs	https://doi.org/10.1002/anie.202016070
Publication status	Published - 14 Jun 2021
Externally published	Yes

Keywords

cell free protein expression
eucaryotic protein expression
Forbidden Coherence Transfer
methyl labeled leucine
methyl TROSY

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10.1002/anie.202016070

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Dubey, A., Stoyanov, N., Viennet, T., Chhabra, S., Elter, S., Borggräfe, J., Viegas, A., Nowak, R. P., Burdzhiev, N., Petrov, O., Fischer, E. S., Etzkorn, M., Gelev, V., & Arthanari, H. (2021). Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems. Angewandte Chemie - International Edition, 60(25), 13783-13787. https://doi.org/10.1002/anie.202016070

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title = "Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems",

abstract = "Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.",

keywords = "cell free protein expression, eucaryotic protein expression, Forbidden Coherence Transfer, methyl labeled leucine, methyl TROSY",

author = "Abhinav Dubey and Nikolay Stoyanov and Thibault Viennet and Sandeep Chhabra and Shantha Elter and Jan Borggr{\"a}fe and Aldino Viegas and Nowak, {Rados{\l}aw P.} and Nikola Burdzhiev and Ognyan Petrov and Fischer, {Eric S.} and Manuel Etzkorn and Vladimir Gelev and Haribabu Arthanari",

note = "Publisher Copyright: {\textcopyright} 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH",

year = "2021",

month = jun,

day = "14",

doi = "10.1002/anie.202016070",

language = "English",

volume = "60",

pages = "13783--13787",

journal = "Angewandte Chemie - International Edition",

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Dubey, A, Stoyanov, N, Viennet, T, Chhabra, S, Elter, S, Borggräfe, J, Viegas, A, Nowak, RP, Burdzhiev, N, Petrov, O, Fischer, ES, Etzkorn, M, Gelev, V & Arthanari, H 2021, 'Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems', Angewandte Chemie - International Edition, vol. 60, no. 25, pp. 13783-13787. https://doi.org/10.1002/anie.202016070

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems. / Dubey, Abhinav; Stoyanov, Nikolay; Viennet, Thibault et al.
In: Angewandte Chemie - International Edition, Vol. 60, No. 25, 14.06.2021, p. 13783-13787.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems

AU - Dubey, Abhinav

AU - Stoyanov, Nikolay

AU - Viennet, Thibault

AU - Chhabra, Sandeep

AU - Elter, Shantha

AU - Borggräfe, Jan

AU - Viegas, Aldino

AU - Nowak, Radosław P.

AU - Burdzhiev, Nikola

AU - Petrov, Ognyan

AU - Fischer, Eric S.

AU - Etzkorn, Manuel

AU - Gelev, Vladimir

AU - Arthanari, Haribabu

PY - 2021/6/14

Y1 - 2021/6/14

N2 - Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.

AB - Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.

KW - cell free protein expression

KW - eucaryotic protein expression

KW - Forbidden Coherence Transfer

KW - methyl labeled leucine

KW - methyl TROSY

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U2 - 10.1002/anie.202016070

DO - 10.1002/anie.202016070

M3 - Journal article

C2 - 33768661

AN - SCOPUS:85105135230

SN - 1433-7851

VL - 60

SP - 13783

EP - 13787

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

IS - 25

ER -

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems

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