Characterization of membrane protein non-native states. 2. the SDS-unfolded states of rhodopsin

Arpana Dutta, Tai Yang Kim, Martina Moeller, Jenny Wu, Ulrike Alexiev, Judith Klein-Seetharaman

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Little is known about the molecular nature of residual structure in unfolded states of membrane proteins. A screen of chemical denaturants to maximally unfold the mammalian membrane protein and prototypic G protein coupled receptor rhodopsin, without interference from aggregation, described in an accompanying paper (DOI 10.1021/bi100338e), identified sodium dodecyl sulfate (SDS), alone or in combination with other chemicals, as the most suitable denaturant. Here, we initiate the biophysical characterization of SDS-denatured states of rhodopsin. Using absorption, steady-state and time-resolved fluorescence spectroscopy, dynamic light scattering, and cysteine accessibility studies, tertiary structure of denatured states was characterized. In agreement with the pattern of secondary structure changes detected by circular dichroism described in the accompanying paper (DOI 10.1021/bi100338e), tertiary structure changes are distinct over four SDS concentration ranges based on the expected predominant micellar structures. Dodecyl maltoside (DM)/SDS mixed micelle spheres (0.05-0.3% SDS) turn into SDS spheres (0.3-3% SDS) that gradually (3-15% SDS) become cylindrical (above 15% SDS). Denatured states in SDS spheres and cylinders show a relatively greater burial of cysteine and tryptophan residues and are more compact as compared to the states observed in mixed micellar structures. Protein structural changes at the membrane/water interface region are most prominent at very low SDS concentrations but reach transient stability in the compact conformations in SDS spheres. This is the first experimental evidence for the formation of a compact unfolding intermediate state with flexible surface elements in a membrane protein.

Original languageEnglish (US)
Pages (from-to)6329-6340
Number of pages12
JournalBiochemistry
Volume49
Issue number30
DOIs
StatePublished - Aug 3 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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