Dual roles of DMPC and CHAPS in the refolding of bacterial opsins in vitro.
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 alpha-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 I1 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 conformation change in bacterial opsins, which led to denaturation. DMPC dependence of chromophore regeneration and the maintenance of the retinal binding pocket suggested that retinal binding pocket formation was part of the large structure changes during stable apoprotein formation.
Sugiyama
Y
Y
Department of Biology, School of Science, Nagoya University, Aichi. ysugiya@bio.nagoya-u.ac.jp
Mukohata
Y
Y
D001059:Apoproteins / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002793:Cholic Acids; D002942:Circular Dichroism; D003902:Detergents; D004134:Dimyristoylphosphatidylcholine; D006217:Halobacterium / Q000378:metabolism*; D010860:Pigments, Biological / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012172:Retinaldehyde; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D002793:Cholic Acids; D003902:Detergents; D010860:Pigments, Biological; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012172:Retinaldehyde; D004134:Dimyristoylphosphatidylcholine