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A proton-detected 4D solid-state NMR experiment for protein structure determination.

Owing to recent advances in instrumentation, methodology and sample-preparation techniques, solid-state NMR spectroscopy is providing unique insights into biological structures at atomic resolution. Three-dimensional structures of proteins and other biological macromolecules can now be determined that are difficult to characterize by X-ray diffraction or solution NMR. Still, the task remains demanding and new methodological developments are needed to make the structure determination more reliable. While it has been demonstrated that assignments are feasible for proteins with over 200 residues, the structure-determination step remains difficult, mainly because spectral overlap introduces ambiguities in restraint assignments during the process of structure generation. These ambiguities can in some cases be resolved, but remain a potential source of errors. Herein, we present an experimental approach that combines the efficient measurement of longrange proton–proton distances in sparsely isotope-labelled samples with proton-detected 3D and 4D correlation spectroscopy. We demonstrate the method in the context of structure determination of the 76-amino-acid protein ubiquitin. Sparsely distributed protons in an otherwise perdeuterated protein yield highly resolved H spectra. The corresponding samples are produced either by expressing perdeuterated protein followed by H back exchange at about 30% of the exchangeable sites or by using suitable precursors to label exclusively a methyl group of alanine, isoleucine, valine or leucine during protein expression. Under fast magic-angle spinning (55 kHz), coherences in such spin systems are sufficiently long-lived to allow not only dipolar, but also scalar-coupling based polarization transfer. H detection increases the sensitivity by a factor of 8 compared to C detection. Similar deuteration schemes have been exploited in solution-state NMR to increase resolution and sensitivity, and to collect precise NOE restraints. An additional advantage arises in the solid phase. In such samples, even the closest H neighbour often corresponds to a long-range contact and dipolar truncation is not an issue, allowing highly-efficient first-order dipolar-recoupling experiments to be used to obtain distance information. Here we use the dipolar recoupling enhanced by amplitude modulation (DREAM) mixing scheme (Figure 1) that provides particularly high polarization-transfer efficiencies due to its adiabatic nature.