We report the crystal structure of a bromide-bound form of the D85S mutant of bacteriorhodopsin, bR(D85S), a protein that uses light energy rather than ATP to pump halide ions across the cell membrane. Comparison of the structure of the halide-bound and halide-free states reveals that both displacements of individual side-chain positions and concerted helical movements occur on the extracellular side of the protein. Analysis of these structural changes reveals how this ion pump first facilitates ion uptake deep within the cell membrane and then prevents the backward escape of ions later in the pumping cycle. Together with the information provided by structures of intermediate states in the bacteriorhodopsin photocycle, this study also suggests the overall design principles that are necessary for ion pumping.
The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.
The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff
base binding site and the structure of the anion-free protein have been obtained in the same crystal form.
Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to
bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main
structural change that accommodates different anions is the repositioning of the polar side chain of S85.
On the basis of these X-ray crystal structures, the prediction is then made that the D85S/D212N double
mutant might bind similar anions and do so over a broader pH range than does the single mutant.
Experimental comparison of the dissociation constants, K
d, for a variety of anions confirms this prediction
and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.
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