Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

We present an approach that speeds up protein solid-state NMR (SSNMR) by 5–20 fold by using paramagnetic doping to condense data-collection time (to ~0.2 s/scan), overcoming a long-standing limitation on slow recycling due to intrinsic 1H T1 longitudinal spin relaxation. By employing low-power schemes under magic-angle spinning at 40 kHz, we show that two-dimensional 13C/13C and 13C/15N SSNMR spectra can be attained for several to tens of nano-moles of β-amyloid fibrils and ubiquitin in just 1–2 days.

We discuss a simple approach to enhance sensitivity for 13 C high-resolution solid-state NMR for proteins in microcrystals by reducing 1 H T 1 relaxation times with paramagnetic relaxation reagents. It was shown that 1 H T 1 values can be reduced from 0.4-0.8 s to 60-70 ms for ubiquitin and lysozyme in D 2 O in the presence of 10 mM Cu(II)Na 2 EDTA without substantial degradation of the resolution in 13 C CPMAS spectra. Faster signal accumulation using the shorter 1 H T 1 attained by paramagnetic doping provided sensitivity enhancements of 1.4-2.9 for these proteins, reducing the experimental time for a given signal-to-noise ratio by a factor of 2.0-8.4. This approach presented here is likely to be applicable to various other proteins in order to enhance sensitivity in 13 C high-resolution solidstate NMR spectroscopy.

Recent developments in sample rotation technology have had a profound impact on magic-angle-spinning NMR. First, rotation frequencies approaching, and even exceeding, strong homonuclear spin interactions have made high-resolution solid-state (1)H spectroscopy much more accessible. Second, the new concept of fast rotation sweep spectroscopy has emerged. Third, high-resolution NMR at cryogenic temperatures has become feasible, offering an enormous sensitivity gain and the opportunity to study a wide range of physical phenomena.

NMR spectroscopy is a prime technique for characterizing atomic-resolution structures and dynamics of biomolecular complexes but for such systems faces challenges of sensitivity and spectral resolution. We demonstrate that the application of (1)H-detected experiments at magic-angle spinning frequencies of >50 kHz enables the recording, in a matter of minutes to hours, of solid-state NMR spectra suitable for quantitative analysis of protein complexes present in quantities as small as a few nanomoles (tens of micrograms for the observed component). This approach enables direct structure determination and quantitative dynamics measurements in domains of protein complexes with masses of hundreds of kilodaltons. Protein-protein interaction interfaces can be mapped out by comparison of the chemical shifts of proteins within solid-state complexes with those of the same constituent proteins free in solution. We employed this methodology to characterize a >300 kDa complex of GB1 with full-length human immunoglobulin, where we found that sample preparation by simple precipitation yields spectra of exceptional quality, a feature that is likely to be shared with some other precipitating complexes. Finally, we investigated extensions of our methodology to spinning frequencies of up to 100 kHz.