Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants

Arpana Dutta, Kalyan C. Tirupula, Ulrike Alexiev, Judith Klein-Seetharaman

Research output: Contribution to journalArticlepeer-review

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 1H 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.

Original languageEnglish (US)
Pages (from-to)6317-6328
Number of pages12
JournalBiochemistry
Volume49
Issue number30
DOIs
StatePublished - Aug 3 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants'. Together they form a unique fingerprint.

Cite this