High Resolution 1H-Detected Solid-State NMR Spectroscopy of Protein Aliphatic Resonances: Access to Tertiary Structure Information

摘要

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely 13C,13C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-13C glycerol or [2]-13C glycerol to dilute the 13C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the 13C or 15N detected version of the experiment. We report here a new labeling scheme, which enables 1H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic 1H resonances. We show further that 13C resolved 3D-1H,1H correlation experiments yield access to long-range proton−proton distances in the protein.