期刊
JOURNAL OF BIOMOLECULAR NMR
卷 58, 期 1, 页码 37-47出版社
SPRINGER
DOI: 10.1007/s10858-013-9802-2
关键词
Magic angle spinning; Solid-state NMR; Paramagnetic relaxation enhancement; Proton detection; Membrane proteins; Condensed data collection
资金
- Natural Sciences and Engineering Research Council of Canada
- Canada Foundation for Innovation
- Ontario Ministry of Economic Development and Innovation
- NSERC PGS fellowship
Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the study of membrane proteins in membrane mimetic and native membrane environments. These experiments often suffer from low sensitivity, due in part to the long recycle delays required for magnetization and probe recovery, as well as detection of low gamma nuclei. In ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work we investigate the sensitivity of C-13 and H-1 detected experiments applied to 27 kDa membrane proteins reconstituted in lipids and packed in small 1.3 mm MAS NMR rotors. We demonstrate that spin diffusion is sufficient to uniformly distribute paramagnetic relaxation enhancement provided by either covalently bound or dissolved CuEDTA over 7TM alpha helical membrane proteins. Using paramagnetic enhancement and low power decoupling in carbon detected experiments we can recycle experiments similar to 13 times faster than under traditional conditions. However, due to the small sample volume the overall sensitivity per unit time is still lower than that seen in the 3.2 mm probe. Proton detected experiments, however, showed increased efficiency and it was found that the 1.3 mm probe could achieve sensitivity comparable to that of the 3.2 mm in a given amount of time. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time.
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