title: Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants.
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 (GuHCl), 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 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, 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.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Arpana
authors.initials: A
authors.lastname: Dutta
authors.forename: Kalyan C
authors.initials: KC
authors.lastname: Tirupula
authors.forename: Ulrike
authors.initials: U
authors.lastname: Alexiev
authors.forename: Judith
authors.initials: J
authors.lastname: Klein-Seetharaman
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding; D055503:Protein Multimerization / Q000187:drug effects*; D012243:Rhodopsin / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014269:Trifluoroacetic Acid / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D012243:Rhodopsin; D014269:Trifluoroacetic Acid; D019791:Guanidine