Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump.

Structural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild-type bR could be a hydroxyl-ion pump. A position between the protonated Schiff base and residue 85 serves as an anion-binding site in the mutant protein, and hydroxyl ions should have access to this site during the O-intermediate of the wild-type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier.

Deprotonation of D96 in bacteriorhodopsin opens the proton uptake pathway.

Despite extensive investigation, the precise mechanism controlling the opening of the cytoplasmic proton uptake pathway in bacteriorhodopsin (bR) has remained a mystery. From an analysis of the X-ray structure of the D96G/F171C/F219L triple mutant of bR and 60 independent molecular dynamics simulations of bR photointermediates, we report that the deprotonation of D96, a key residue in proton transfer reactions, serves two roles that occur sequentially. First, D96 donates a proton to the Schiff base. Subsequently, the deprotonation of D96 serves to "unlatch" the cytoplasmic side. The latching function of D96 appears to be remarkably robust, functioning to open hydration channels in all photointermediate structures. These results suggest that the protonation state of D96 may be the critical biophysical cue controlling the opening and closing of the cytoplasmic half-channel in bR. We suspect that this protonation-switch mechanism could also be utilized in other proton pumps to minimize backflow and reinforce directionality.

Membrane-protein stability in a phospholipid-based crystallization medium.

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.

Crystal structure of the bromide-bound D85S mutant of bacteriorhodopsin: principles of ion pumping.

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.

Specificity of anion binding in the substrate pocket of bacteriorhodopsin.

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.

Crystallization of membrane proteins from media composed of connected-bilayer gels.

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.