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 stereoselective 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.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…Altogether, 13 C-edited experiments might have been underexploited for in-cell structural biology by NMR. 13 C-labeling is most often more expensive than 15 N-labeling, but the advantages of 2D 1 H– 13 C correlation spectra show promising results, which could be further improved by some recent isotope labeling methods and pulse sequences. , …”

“…Introducing partially deuterated isotope-labeled amino acids would probably help to improve in-cell NMR sensitivity. 111,438 While in situ production can accommodate a number of isotope-labeling schemes in E. coli, there are a number of labeling possibilities to explore in insect and mammalian cells. These might also permit to use 13 C-direct detection, which appears to be necessary to characterize disordered proteins at physiological temperatures, 178,436 together with and 1 Ha- 13 Ca correlations.…”

“…One-dimensional spectra can yield equivalent information using amino acid-type specific 15 N-labeling at about twice lower concentrations. A number of strategies shall be combined to improve sensitivity: better isotope-labeling schemes, notably when introducing deuteration, better pulse sequences adapted to the cellular viscosity, paramagnetic T1 relaxation enhancement, − continuous readout using time-resolved nonuniform sampling, ,− deep-learning assisted spectral reconstruction, − denoising procedures, ,− deep-learning assisted peak picking, etc. Using all these advanced techniques, it might be possible to record exploitable in-cell 2D NMR spectra of proteins/nucleic acids at intracellular concentrations of a few μmol/L.…”

“…Uniform 15 N-labeling provides rich structural information, but might be limited in terms of sensitivity in the long term. Introducing partially deuterated isotope-labeled amino acids would probably help to improve in-cell NMR sensitivity. , While in situ production can accommodate a number of isotope-labeling schemes in E. coli , there are a number of labeling possibilities to explore in insect and mammalian cells. These might also permit to use 13 C-direct detection, which appears to be necessary to characterize disordered proteins at physiological temperatures, , together with 1 Hα- 13 Cα correlations. − Low-γ nuclei detection may actually be adapted to the slower tumbling times observed in cells, notably in absence of uniform deuteration.…”

Bacterial Transcriptional Regulators: A Road Map for Functional, Structural, and Biophysical Characterization

“…However, NMR as a structural technique is inherently limited by concentration, protein size, and the existence of highly disordered regions. In this context, the development of new NMR methodologies continues to drive the field of functional dynamics forward, with contributions to new isotope labeling strategies [143] and pulse sequences [144]. The field has taken advantage of a combination of deuteration and TROSY-based [145] triple resonance experiments [146], which has allowed for the measurement of good-quality NMR spectra from other TRs in different ligation states by avoiding fast relaxation.…”