Crystal structure of the L intermediate of bacteriorhodopsin: evidence for vertical translocation of a water molecule during the proton pumping cycle

For structural investigation of the L intermediate of bacteriorhodopsin, a 3D crystal belonging to the space group P622 was illuminated with green light at 160 K and subsequently with red light at 100 K. This yielded a approximately 1:4 mixture of the L intermediate and the ground-state. Diffraction data from such crystals were collected using a low flux of X-rays ( approximately 2 x 10(15) photons/mm2 per crystal), and their merged data were compared with those from unphotolyzed crystals. These structural data, together with our previous data, indicate that the retinal chromophore, which is largely twisted in the K-intermediate, takes a more planar 13-cis, 15-anti configuration in the L intermediate. This configurational change, which is accompanied by re-orientation of the Schiff base N-H bond towards the intracellular side, is coupled with a large rotation of the side-chain of an amino acid residue (Leu93) making contact with the C13 methyl group of retinal. Following these motions, a water molecule, at first hydrogen-bonded to the Schiff base and Asp85, is dragged to a space that is originally occupied by Leu93. Diffraction data from a crystal containing the M intermediate showed that this water molecule moves further towards the intracellular side in the L-to-M transition. It is very likely that detachment of this water molecule from the protonated Schiff base causes a significant decrease in the pKa of the Schiff base, thereby facilitating the proton transfer to Asp85. On the basis of these observations, we argue that the vertical movement of a water molecule in the K-to-L transition is a key event determining the directionality of proton translocation in the protein.     

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.     

Structure of bacteriorhodopsin at 1.55 A resolution.

Th?e atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K,L, M1, M2, and M2' intermediates of the photocycle

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.     

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK(a) of the Schiff base (the primary proton donor) and the low pK(a) of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Water dynamics simulation as a tool for probing proton transfer pathways in a heptahelical membrane protein

The proton transfer pathway in a heptahelical membrane protein, the light-driven proton pump bacteriorhodopsin (BR), is probed by a combined approach of structural analysis of recent X-ray models and molecular dynamics (MD) simulations that provide the diffusion pathways of internal and external water molecules. Analyzing the hydrogen-bond contact frequencies of the water molecules with protein groups, the complete proton pathway through the protein is probed. Beside the well-known proton binding sites in the protein interior-the protonated Schiff base, Asp85 and Asp96, and the H(5)O(2) (+) complex stabilized by Glu204 and Glu194-the proton release and uptake pathways to the protein surfaces are described in great detail. Further residues were identified, by mutation of which the proposed pathways can be verified. In addition the diffusion pathway of water 502 from Lys216 to Asp96 is shown to cover the positions of the intruding waters 503 and 504 in the N-intermediate. The transiently established water chain in the N-state provides a proton pathway from Asp96 to the Schiff base in the M- to N-transition in a Grotthus-like mechanism, as concluded earlier from time-resolved Fourier transform infrared experiments [le Coutre et al., Proc Nat Acad Sci USA 1995;92:4962-4966].     

Transducer protein HtrI controls proton movements in sensory rhodopsin I

Sensory rhodopsin I (SR-I lambda(max) 587 nm) is a phototaxis receptor in the archaeon Halobacterium salinarium. Photoisomerization of retinal in SR-I generates a long-lived intermediate with lambda(max) 373 nm which transmits a signal to the membrane-bound transducer protein HtrI. Although SR-I is structurally similar to the electrogenic proton pump bacteriorhodopsin (BR), early studies showed its photoreactions do not pump protons, nor result in membrane hyperpolarization. These studies used functionally active SR-I, that is, SR-I complexed with its transducer HtrI. Using recombinant DNA methods we have expressed SR-I protein containing mutations in ionizable residues near the protonated Schiff base, and studied wild-type and site-specifically mutated SR-I in the presence and absence of the transducer protein. UV-Vis kinetic absorption spectroscopy, FT-IR, and pH and membrane potential probes reveal transducer-free SR-I photoreactions result in vectorial proton translocation across the membrane in the same direction as that of BR. This proton pumping is suppressed by interaction with transducer which diverts the proton movements into an electroneutral path. A key step in this diversion is that transducer interaction raises the pK(a) of the aspartyl residue in SR-I (Asp76) which corresponds to the primary proton-accepting residue in the BR pump (Asp85). In transducer-free SR-I, our evidence indicates the pK(a) of Asp76 is 7.2, and ionized Asp76 functions as the Schiff base proton acceptor in the SR-I pump. In the SR-I/HtrI complex, the pK(a) of Asp76 is 8.5, and therefore at physiological pH (7.4) Asp76 is neutral. Protonation changes on Asp76 are clearly not required for signaling since the SR-I mutants D76N and D76A are active in phototaxis. The latent proton-translocation potential of SR-I may reflect the evolution of the SR-I sensory signaling mechanism from the proton pumping mechanism of BR.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump

High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. In the extracellular region an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline kink. The bulge is stabilized by hydrogen bonding of the main chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.     

Chromophore-protein-water interactions in the L intermediate of bacteriorhodopsin: FTIR study of the photoreaction of L at 80 K.

Using FTIR spectroscopy, perturbations of several residues and internal water molecules have been detected when light transforms all-trans bacteriorhodopsin (BR) to its L intermediate having a 13-cis chromophore. Illumination of L at 80 K results in an intermediate L' absorbing around 550 nm. L' thermally converts to the original BR only at >130 K. In this study, we used the light-induced transformation of L to L' at 80 K to identify some amino acid residues and water molecules that closely interact with the chromophore and distinguish them from those residues not affected by the photoreaction. The L minus L' FTIR difference spectrum shows that the chromophore in L' is in the all-trans configuration. The perturbed states of Asp96 and Val49 and of the environment along the aliphatic part of the retinal and Lys216 seen in L are not affected by the L --> L' photoreaction. On the other hand, the environments of the Schiff base of the chromophore, of Asp115, and of water molecules close to Asp85 returned in L' to their state in which they originally had existed in BR. The water molecules that are affected by the mutations of Thr46 and Asp96 also change to a different state in the L --> L' transition, as indicated by transformation of a water O-H vibrational band at 3497 cm-1 in L into an intense peak at 3549 cm-1 in L'. Notably, this change of water bands in the L --> L' transition at 80 K is entirely different from the changes observed in the BR --> K photoreaction at the same temperature, which does not show such intense bands. These results suggest that these water molecules move closer to the Schiff base as a hydrogen bonding cluster in L and L', presumably to stabilize its protonated state during the BR to L transition. They may contribute to the structural constraints that prevent L from returning to the initial BR upon illumination at 80 K.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

Strongly hydrogen-bonded water molecules in the Schiff base region of rhodopsins.

In many rhodopsins, a positively charged retinal chromophore is stabilized by a negatively charged carboxylate, and the presence of bound water molecules has been found in the Schiff base region by X-ray crystallography of various rhodopsins. Low-temperature Fourier-transform infrared (FTIR) spectroscopy can directly monitor hydrogen-bonding alterations of internal water molecules of rhodopsins. In particular, we found that a bridged water molecule between the Schiff base and Asp 85 in bacteriorhodopsin (BR), a light-driven proton-pump protein, forms an extremely strong hydrogen bond. It is likely that a hydration switch of the water from Asp 85 to Asp 212 plays an important role in the proton transfer in the Schiff base region of BR. Comprehensive studies of archaeal and visual rhodopsins have revealed that strongly hydrogen-bonded water molecules are only found in the proteins exhibiting proton-pump activities. Strongly hydrogen-bonded water molecules and its transient weakening may be essential for the proton-pump function of rhodopsins.     

The protonation-deprotonation kinetics of the protonated Schiff base in bicelle bacteriorhodopsin crystals

In the recently published x-ray crystal structure of the "bicelle" bacteriorhodopsin (bbR) crystal, the protein has quite a different structure from the native and the in cubo bacteriorhodopsin (cbR) crystal. Instead of packing in parallel trimers as do the native membrane and the cbR crystals, in the bbR crystal the protein packs as antiparallel monomers. To date, no functional studies have been performed, to our knowledge, to investigate if the photocycle is observed in this novel protein packing structure. In this study, both Raman and time-resolved transient absorption spectroscopy are used to both confirm the presence of the photocycle and investigate the deprotonation-reprotonation kinetics of the Schiff base proton in the bbR crystal. The observed rates of deprotonation and reprotonation processes of its Schiff base have been compared to those observed for native bR under the same conditions. Unlike the previously observed similarity of the rates of these processes for cbR crystals and those for native bacteriorhodopsin (bR), in bbR crystals the rate of deprotonation has increased by 300%, and the rate of reprotonation has decreased by nearly 700%. These results are discussed in light of the changes observed when native bR is delipidated or monomerized by detergents. Both the change of the hydrophobicity of the environment around the protonated Schiff base and Asp85 and Asp96 (which could change the pKa values of proton donor-acceptor pairs) and the water structure in the bbR crystal are offered as possible explanations for the different observations.     

Structural characterization of the L-to-M transition of the bacteriorhodopsin photocycle.

Structural intermediates occurring in the photocycle of wild-type bacteriorhodopsin are trapped by illuminating hydrated, glucose-embedded purple membrane at 170 K, 220 K, 230 K, and 240 K. We characterize light-induced changes in protein conformation by electron diffraction difference Fourier maps, and relate these to previous work on photocycle intermediates by infrared (FTIR) spectroscopy. Samples illuminated at 170 K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm resolution limit of our data. Difference maps obtained with samples illuminated at 220 K, 230 K, and 240 K, respectively, reveal a progressively larger structural response in helix F when the protein is still in the M state, as judged by the FTIR spectra. Consistent with previous structural studies, an adjustment in the position or in the degree of ordering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin experiences minimal change in protein structure until a proton is transferred from the Schiff base to Asp85. The M intermediate then undergoes a conformational evolution that opens a hydrated "half-channel," allowing the subsequent reprotonation of the Schiff base by Asp96.     

Crystal structure of the M intermediate of bacteriorhodopsin: allosteric structural changes mediated by sliding movement of a transmembrane helix

Structural changes in the proton pumping cycle of wild-type bacteriorhodopsin were investigated by using a 3D crystal (space group P622)prepared by the membrane fusion method. Protein-protein contacts in the crystal elongate the lifetime of the M intermediate by a factor of approximately 100,allowing high levels of the M intermediate to accumulate under continuous illumination. When the M intermediate generated at room temperature was exposed to a low flux of X-rays (approximately 10(14) photons/mm2), this yellow intermediate was converted into a blue species having an absorption maximum at 650 nm. This color change is suggested to accompany a configuration change in the retinal-Lys216 chain. The true conformational change associated with formation of the M intermediate was analyzed by taking the X-radiation-induced structural change into account. Our result indicates that, upon formation of the M intermediate, helix G move stowards the extra-cellular side by, on average, 0.5 angstroms. This movement is coupled with several reactions occurring at distal sites in the protein: (1) reorientation of the side-chain of Leu93 contacting the C13 methyl group of retinal, which is accompanied by detachment of a water molecule from the Schiff base; (2) a significant distortion in the F-G loop, triggering destruction of a hydrogen bonding interaction between a pair of glutamate groups (Glu194 and Glu204); (3) formation of a salt bridge between the carboxylate group of Glu204 and the guanidinium ion of Arg82, which is accompanied by a large distortion in the extra-cellular half of helix C; (4)noticeable movements of the AB loop and the cytoplasmic end of helix B. But, no appreciable change is induced in the peptide backbone of helices A,D, E and F. These structural changes are discussed from the viewpoint of translocation of water molecules.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane.

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.