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000013097 0247_ $$2DOI$$a10.1021/bi100338e
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000013097 084__ $$2WoS$$aBiochemistry & Molecular Biology
000013097 1001_ $$0P:(DE-HGF)0$$aDutta, A.$$b0
000013097 245__ $$aCharacterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants
000013097 260__ $$aColumbus, Ohio$$bAmerican Chemical Society$$c2010
000013097 300__ $$a6317 - 6328
000013097 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article
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000013097 440_0 $$0798$$aBiochemistry$$v49$$x0006-2960$$y30
000013097 500__ $$aThis work was in part supported by National Science Foundation CAREER Grant CC044917, National Institutes of Health Grant NLM108730, and the Pennsylvania Department of Health.
000013097 520__ $$aLittle is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHC1), and trifluoroacetic acid (TFA) on rhodopsin's secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS PAGE, dependence of ellipticity on protein concentration, and ID H-1 NMR we show that aggregates form in the presence of GuHCI, TEA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.
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000013097 7001_ $$0P:(DE-HGF)0$$aTirupula, K.C.$$b1
000013097 7001_ $$0P:(DE-HGF)0$$aAlexiev, U.$$b2
000013097 7001_ $$0P:(DE-Juel1)VDB44599$$aKlein-Seetharaman, J.$$b3$$uFZJ
000013097 773__ $$0PERI:(DE-600)1472258-6$$a10.1021/bi100338e$$gVol. 49, p. 6317 - 6328$$p6317 - 6328$$q49<6317 - 6328$$tBiochemistry$$v49$$x0006-2960$$y2010
000013097 8567_ $$uhttp://dx.doi.org/10.1021/bi100338e
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000013097 9141_ $$y2010
000013097 9131_ $$0G:(DE-Juel1)FUEK505$$aDE-HGF$$bSchlüsseltechnologien$$kP45$$lBiologische Informationsverarbeitung$$vBioSoft: Makromolekulare Systeme und biologische Informationsverarbeitung$$x0
000013097 9132_ $$0G:(DE-HGF)POF3-551$$1G:(DE-HGF)POF3-550$$2G:(DE-HGF)POF3-500$$aDE-HGF$$bKey Technologies$$lBioSoft  Fundamentals for future Technologies in the fields of Soft Matter and Life Sciences$$vFunctional Macromolecules and Complexes$$x0
000013097 9201_ $$0I:(DE-Juel1)ISB-2-20090406$$d31.12.2010$$gISB$$kISB-2$$lMolekulare Biophysik$$x0
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