Structural changes of purple membrane and bacteriorhodopsin during its denaturation induced by high pH

Bacteriorhodopsin (bR) trimers naturally form two-dimensional hexagonal crystals in purple membrane (PM), which make it very stable. However, the dnaturation of bR was found to occur during a very narrow pH range when the pH was increased above 12.0, as indicated by inactivation of the photochemical cycle observed by flash photolysis kinetic spectra. Here, atomic force microscopy was used to study the surface structural changes of PM during the denaturation process induced by high pH. Together with the absorption and fluorescence spectra, it was found that the structural changes could be divided into three steps. First, some hydrophobic amino acids of bR become exposed to the aqueous environment and PM loses its 2D crystalline structure, transforming into the so-called "nonisland" structure. Second, bR molecules are extracted out of membrane and form protrusions on the surface like islands in the sea; therefore, the "nonisland" structure transforms into the "island" structure. Finally, most bRs break off from the membrane and form large depositions.