Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins

¹H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using ²H excitation instead of ¹H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T₁, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient ²H-excitation and ²H-¹³C cross-polarization (CP) step, combined with ¹H detection. We show that by using ²H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H₂O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties. Proton-detected magic-angle spinning solid-state NMR spectroscopy using deuterated proteins facilitates structural biology. Solid deuterated proteins that cannot be unfolded/refolded to exchange the deuterons back with protons and proteins that can only be studied in their native environments show a low intrinsic sensitivity in NMR experiments. For such systems, initial excitation of the deuterons can be very useful.