Difference Fourier analysis of "surface features" of bacteriorhodopsin using glucose-embedded and frozen-hydrated...

A difference Fourier map of the projected structure of bacteriorhodopsin has been synthesized from electron diffraction amplitudes collected from membranes prepared in the glucose-embedded state and the frozen-hydrated state. Phases of a recently published data set for glucose-embedded specimens were used for the difference Fourier map. Moderate resolution (9 A) and high resolution (4.25 A) maps both indicate that glucose is exchangeable for water in the region of the map corresponding to the lipid regions. We interpret this as indicating that there is a small surface depression in this region of the structure. The depth of this feature is estimated to be 1/6 the thickness of the protein region in the membrane. The data obtained in this study rules out the existence of an aqueous transmembrane channel, the dimensions of which are large enough to allow free exchange of glucose for water. Several new features are also observed in the protein region of the membrane. These features are probably due to segments of the polypeptide at the aqueous interface that are well ordered in frozen-hydrated specimens but not in glucose-embedded specimens. Candidate structures for the origin of these features are extensions of the helices, or linker regions connecting the helices.