Formation Of A Core In SDS Denatured States Of Rhodopsin

Abstract

A general understanding of the folding pathway of helical membrane proteins has remained elusive due to the limited number of membrane proteins investigated to date. In order to begin to establish conditions under which the folding pathway of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied, we have measured its secondary and tertiary structure changes under chemical denaturing conditions. A significant decrease in ellipticity at 222nm of maximally 40% is observed on adding sodium dodecyl sulfate (SDS) as a denaturant suggesting unfolding of the native helices. Tertiary structure is disrupted already at very low SDS concentrations of 0.05%, as evidenced by the loss of retinal chromophore, increase in tryptophan fluorescence and increase in cysteine accessibility. However, at intermediate SDS concentrations (0.1% to 3%), there is a decrease in tryptophan fluorescence accompanied by a simultaneous decrease in cysteine reactivity indicating formation of a compact intermediate. Light-scattering confirms that this decrease is not due to unspecific aggregation of the protein. Site-directed mutagenesis indicates that the reactive cysteines in this intermediate are located in the cytoplasmic domain of rhodopsin. At high SDS concentrations, between 7% and 15%, where a large amount of additional helix is disrupted, there is an increase in tryptophan fluorescence and cysteine reactivity. However, with further increase in SDS concentration up to 30%, there is a drop in fluorescence and cysteine accessibility suggesting formation of a core in this largely unfolded state. Existence of a folding core during early stages of folding of rhodopsin has also been postulated by the long-range interaction model and has been predicted by earlier computational experiments.