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 ¹³ C-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: Forbidden Coherence Transfer; cell free protein expression; eucaryotic protein expression; methyl TROSY; methyl labeled leucine.