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 newspaperJournal articleResearchpeer-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 languageEnglish
JournalAngewandte Chemie - International Edition
Volume60
Issue25
Pages (from-to)13783-13787
Number of pages5
ISSN1433-7851
DOIs
Publication statusPublished - 14 Jun 2021
Externally publishedYes

Keywords

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

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