The use of amphipols for NMR structural characterization of 7-TM proteins.
The use of amphipols for NMR structural characterization of 7-TM proteins.
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DOI：
10.1007/s00232-014-9669-5
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发表时间：
2014-10
影响因子：
2.4
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Elter, Shantha;Raschle, Thomas;Arens, Sabine;Viegas, Aldino;Gelev, Vladimir;Etzkorn, Manuel;Wagner, Gerhard
While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labelled GPCRs are still very limited. Here we report on recent progress towards NMR structural studies of the melanocortin-2 and -4 receptor, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical scale refolding experiments on the melanocortin-2 receptor show very similar behavior to results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straight forward and impeded detailed NMR insights for the two GPCRs. While well resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.
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DOI：
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发表时间：
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影响因子：
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DOI：
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发表时间：
2013-03-01
影响因子：
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作者：
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