Technical Highlight - May 2009
Short description: Improved sensitivity for solid-state nuclear magnetic resonance spectroscopy makes this technique useful for large biomolecules.Nature Meth. 6, 215-218 (2009)
NMR spectroscopy is widely used to study proteins. It is mostly employed for proteins in solution, although solid-state NMR is giving exciting results for proteins in crystals and for insoluble proteins in supramolecular complexes such as membranes, virus particles or amyloid fibrils.
But solid-state NMR has been held back by its requirement for relatively large sample amounts (0.5–1 micromolar) to produce spectra with sufficient sensitivity to determine a three-dimensional protein structure. Now, Wickramasinghe et al. 1 reveal a technique that works with a substantially reduced sample concentration and speeds up data collection. They call their technique paramagnetic-relaxation-assisted condensed data collection (PACC).
Part of the problem with data collection in solid-state NMR has been inefficiency due to the long delays that occur while waiting for the magnetization to recover between scans. These 'recycle delays' can account for between 95% and 99% of the experimental time.
Wickramasinghe et al. reduce the 1H longitudinal-relaxation time (T 1) for hydrated proteins by combining paramagnetic doping, very fast magic-angle spinning and fast recycling of low-power radiofrequency pulse sequences. Recycling takes only about 0.2 seconds per scan for hydrated proteins, decreasing data-collection times by between 5- and 20-fold. Under these conditions, small samples of 13C- and 15N-labeled proteins should produce interpretable two-dimensional solid-state NMR spectra.
Ultimately, this technique will be suitable for biomolecules at nanomolar concentrations. For example, it might be useful for determining the three-dimensional structure of membrane proteins, which usually make up only a tiny fraction of the sample. The authors' results with ubiquitin are encouraging, as they obtained a reasonable spectrum in 3 hours when the protein constituted only 2% of the total sample volume.
N. P. Wickramasinghe et al. Nanomole-scale protein solid-state NMR by breaking intrinsic 1H T1 boundaries.
Nature Meth 6, 215-218 (2009). doi:10.1038/nmeth.1300