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 Burdziev,, Ognyan Petrov Eric S. Fischer, Manuel Etzkorn, Vladimir Gelev, Haribabu Arthanari
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.
Methyl-based magnetization usage can strongly benefit investigations of large protein systems via NMR spectroscopy. In this work we present a simple and cost-effective method to obtain locally deuterated leucine suitable for methyl NMR and quantify its benefits. The approach substantially expands the applicability of methyl labeling in emerging expression systems including cell-free and eukaryotic systems.
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