Abstract
The limits of resolution that can be obtained in 1H-15N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee-Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide 1H resonances. Heteronuclear decoupling of 15N from the 1H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly 2H and 15N enriched protein where the amides have been exchanged in normal water, MAS at approximately 20 kHz, and WALTZ-16 decoupling of the 15N nuclei. The combination of these techniques results in average 1H lines of only approximately 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and 15N decoupling are described for achieving the best possible performance in these experiments.
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