Structure of an Early Intermediate in the M-State Phase of the Bacteriorhodopsin Photocycle

AbstractThe structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230K has been determined by x-ray crystallography to a resolution of 2.0Å. Three-dimensional crystals were trapped by illuminating with actinic light at 230K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13—C14 double bond appears to be complete, but a relatively large torsion angle of 26° is still seen for the C14—C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (MN) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics

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

AbstractStructural 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

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{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution