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

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• 作者：Sam Asami ; Peter Schmieder ; Bernd Reif
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：November 3, 2010
• 年：2010
• 卷：132
• 期：43
• 页码：15133-15135
• 全文大小：172K
• 年卷期：v.132,no.43(November 3, 2010)
• ISSN：1520-5126

文摘

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.