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nmrlearner 12-18-2013 04:00 PM

High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning.
 
High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning.

Related Articles High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning.

J Biomol NMR. 2013 Dec 13;

Authors: Ward ME, Wang S, Krishnamurthy S, Hutchins H, Fey M, Brown LS, Ladizhansky V

Abstract
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 (13)C and (1)H 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 ~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.


PMID: 24338448 [PubMed - as supplied by publisher]



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