Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα MAS NMR without re-protonation bottleneck

Abstract

Amide-proton detected magic-angle spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with H2O-based culture medium doped with deuterated cell lysate allow to overcome this "reprotonation bottleneck", while retaining a high level of deuteration (ca. 80 %) and narrow line widths. We quantified coherence life times of several proteins prepared with this labelling pattern over a range of MAS frequencies (40-100 kHz). We demonstrate that under commonly used conditions (50-60 kHz MAS), amide 1H line widths with our labelling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33-50 kDa size range many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and show that such media can be used identically to commercial media. The residual protonation of H sites allows for well-resolved H-detected spectra and H resonance assignment, exemplified by the de novo assignment of 168 H sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm/100 kHz MAS experiments.