Conformational transitions in delipidated and detergent-solubilized bacteriorhodopsin

Abstract

Bacteriorhodopsin in the purple membrane from Halobacterium halobium is a integral protein with a high content of α-helix. It is the most extensively studied of all the integral membrane proteins. Here the bacteriorhodopsin is solubilized from purple membrane with a mild detergent and obtained in a delipidated form. It appears that bacteriorhodopsin in deoxycholate solution retains its three dimensional structure and has the proton pumping activity when it is incorporated into soybean phospholipid vesicles. Delipidated and detergent-solubilized bacteriorhodopsin has somewhat lower transition temperatures at 90-92℃ in comparison to those of purple membrane which appears around 100℃. They were, however, much higher than those shown by the simple soluble proteins, such as ribonuclease and chymotrypsin. At pH of 8.0 or above, the thermal transition profiles are sharp and suggested a cooperative and single-step conformational transition, possibly a α-helix to random coil transition. At pH values below 8.0, the profile show two transitions. The thermal transitions at lower temperature, however, correspond to those of pure detergent, sodium deoxycholate. Therefore the bacteriorhodopsin in this pH region is still likely to have a single conformational transition. The effect of alcohols on the thermal transitions of delipidated and detergentsolubilized bacteriorhodopsin were also studied. There was no appreciable absorbance at 280 nm near room temperature when the bacteriorhodopsin is exposed to a increasing concentration of ethanol despite the results from 540 nm spectra and circular dichroism spectra strongly suggested increased unfolding of α-helical structure of this protein. The absorbance difference going from room temperatures to the unfolded state decreased with the increase in ethanol concentration. A discussion on these phenomena and also the effect of methanol and propanol is given. Thermodynamic parameters of this thermal transition obtained and compared ...