Abstract

The bacterial opsins can be refolded to regenerate the chromophore by transfer from SDS to DMPC/CHAPS/SDS mixed micelles in the presence of retinal. A sequential refolding model has been proposed for bacterioopsin [Booth et al. (1995) Nature Struct Biol. 2,139-143]. However, the roles of DMPC and CHAPS in the refolding process are not clear. In this study we measured the effects of DMPC and CHAPS on the refolding of bacterial opsins in vitro by CD and fluorescence spectroscopy. In contrast to in experiments in the presence of large amounts of DMPC, the process of retinal binding pocket formation was a rate-determining step in overall chromophore regeneration with relatively low concentrations of DMPC. CHAPS triggered a-helix formation and long-range interactions between the helices within 1 s by providing a suitable hydrophobic environment for bacterial opsins. This CHAPS-induced transient molten globule-like structure would be identical to Ii postulated by Booth et al., to which DMPC bound and induced the proper packing of the side chains to form a retinal binding pocket. If DMPC was not present, CHAPS induced another conforma-tion change in bacterial opsins, which led to denaturation. DMPC dependence of chromo-phore regeneration and the maintenance of the retinal binding pocket suggested that retinal