Dehydration-induced molecular structural changes of purple membrane of halobacterium halobium.

The nature and extent of dehydration-induced molecular structural changes of the purple membrane of Halobacterium halobium have been studied by absorption and circular dichroism spectra in solution and in oriented membrane films. High glycerol concentrations, exhaustive dry nitrogen gas flushing, and exhaustive high-vacuum pumping were employed as dehydrants. The effect of these dehydrants on the spectra were reversible, similar, and additive. Analysis of the spectral changes observed at maximal dehydration revealed: (a) at least two additional optical states of the bacteriorhodopsin, one at higher energy and another at lower energy than the characteristic dark- and light-adapted states; (b) no change in the dichroic ratio at the visible absorption maximum within experimental error; (c) no change in the polarity of the visible monomeric retinylidene circular dichroic bands; (d) pronounced reduction in the characteristic excitonic interactions among the retinals in the hexagonal crystalline lattice of the membrane; (e) no changes in the native structural anisotropism of the membrane in respect to the orientation of the amino acid aromatic rings of the bacteriorhodopsin; (f) no changes in the secondary structure of the bacteriorhodopsin; and (g) a net tilting of approximately 20.5 degrees per segment of the helical polypeptide segments of the bacteriorhodopsin away from the membrane normal. A molecular model of the structural changes of the membrane resulting from water removal consistent with these findings can be constructed. Dehydration results in only subtle localized tertiary structural changes of the protein which do not significantly alter its shape or size. However, there are pronounced global supramolecular structural changes of the membrane. Water removal, which is most likely to be from the lipid headgroups of the membrane, disrupts the interactions responsible for maintaining the native crystalline lattice of the membrane resulting in pronounced randomization of the positions of the proteins in the membrane.