Denaturation and renaturation of bacteriorhodopsin in detergents and lipid-detergent mixtures.
The denatured and renatured states of bacteriorhodopsin have been studied in detergents and lipid/detergent mixtures by using ultraviolet and visible light absorption spectroscopy, fluorescence spectroscopy, and circular dichroism. Upon solubilization in sodium dodecyl sulfate, bacteriorhodopsin undergoes denaturation with partial loss of the secondary structure and loss of retinal binding ability. In contrast, delipidated bacteriorhodopsin retains its native structure in deoxycholate and undergoes denaturation only on photobleaching, which results in a partial loss of the secondary structure. Native secondary structure spontaneously reforms upon addition of phospholipid/cholate mixtures to bacterio-opsin denatured in sodium dodecyl sulfate. Upon subsequent addition of retinal, up to 95% of the native chromophore (lambdamax = 550-560 nm) is restored. Renaturation also restores the characteristics of light and dark adaptation and tight retinal binding (Kb greater than 10(7) M-1). Renaturation of delipidated bacteriorhodopsin denatured in sodium dodecyl sulfate also occurs without phospholipid upon the addition of retinal and Triton X-100 or octyl glucoside. The equilibrium between monomeric and oligomeric states of bacteriorhodopsin has been studied by circular dichroism and fluorescence quenching. Bacteriorhodopsin renatured with Triton X-100 or phospholipid/cholate is monomeric. However, removal of the detergents from renatured protein under specific conditions results in formation of an oligomer in which quaternary interactions are regenerated. The application of fluorescence quenching to the determination of oligomeric size in intrinsic membrane proteins is discussed.
D001436:Bacteriorhodopsins / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000494:pharmacology*; D004134:Dimyristoylphosphatidylcholine; D006217:Halobacterium / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011485:Protein Binding; D011489:Protein Denaturation; D012172:Retinaldehyde / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D002338:Carotenoids; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; D012172:Retinaldehyde; D004134:Dimyristoylphosphatidylcholine