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@ARTICLE{Dutta:13097,
      author       = {Dutta, A. and Tirupula, K.C. and Alexiev, U. and
                      Klein-Seetharaman, J.},
      title        = {{C}haracterization of membrane protein non-native states.
                      1. {E}xtent of unfolding and aggregation of rhodopsin in the
                      presence of chemical denaturants},
      journal      = {Biochemistry},
      volume       = {49},
      issn         = {0006-2960},
      address      = {Columbus, Ohio},
      publisher    = {American Chemical Society},
      reportid     = {PreJuSER-13097},
      pages        = {6317 - 6328},
      year         = {2010},
      note         = {This work was in part supported by National Science
                      Foundation CAREER Grant CC044917, National Institutes of
                      Health Grant NLM108730, and the Pennsylvania Department of
                      Health.},
      abstract     = {Little 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.},
      keywords     = {J (WoSType)},
      cin          = {ISB-2},
      ddc          = {570},
      cid          = {I:(DE-Juel1)ISB-2-20090406},
      pnm          = {BioSoft: Makromolekulare Systeme und biologische
                      Informationsverarbeitung},
      pid          = {G:(DE-Juel1)FUEK505},
      shelfmark    = {Biochemistry $\&$ Molecular Biology},
      typ          = {PUB:(DE-HGF)16},
      UT           = {WOS:000280416100004},
      doi          = {10.1021/bi100338e},
      url          = {https://juser.fz-juelich.de/record/13097},
}