Proton Pump Bacteriorhodopsin Research Articles

The Roles of an Extended N-terminal Region and ETD Motif in a Pump-like Cation Channelrhodopsin Discovered in a Lake Microbiome

Channelrhodopsins are light-gated ion channels consisting of seven-transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

Unique hydrogen-bonding network in a viral channelrhodopsin

Channelrhodopsins (CRs) are used as key tools in optogenetics, and novel CRs, either found from nature or engineered by mutation, have greatly contributed to the development of optogenetics. Recently CRs were discovered from viruses, and crystal structure of a viral CR, OLPVR1, reported a very similar water-containing hydrogen-bonding network near the retinal Schiff base to that of a light-driven proton-pump bacteriorhodopsin (BR). In both OLPVR1 and BR, nearly planar pentagonal cluster structures are comprised of five oxygen atoms, three oxygens from water molecules and two oxygens from the Schiff base counterions. The planar pentagonal cluster stabilizes a quadrupole, two positive charges at the Schiff base and an arginine, and two negative charges at the counterions, and thus plays important roles in light-gated channel function of OLPVR1 and light-driven proton pump function of BR. Despite similar pentagonal cluster structures, present FTIR analysis revealed different hydrogen-bonding networks between OLPVR1 and BR. The hydrogen bond between the protonated Schiff base and a water is stronger in OLPVR1 than in BR, and internal water molecules donate hydrogen bonds much weaker in OLPVR1 than in BR. In OLPVR1, the bridged water molecule between the Schiff base and counterions forms hydrogen bonds to D76 and D200 equally, while the hydrogen-bonding interaction is much stronger to D85 than to D212 in BR. The present interpretation is supported by the mutation results, where D76 and D200 equally work as the Schiff base counterions in OLPVR1, but D85 is the primary counterion in BR. This work reports highly sensitive hydrogen-bonding network in the Schiff base region, which would be closely related to each function through light-induced alterations of the network.

Proton Release Reactions in the Inward H+ Pump NsXeR.

Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.

A Proteorhodopsin-Related Photosensor Expands the Repertoire of Structural Motifs Employed by Sensory Rhodopsins.

Microbial rhodopsins are light-activated retinal-binding membrane proteins that perform a variety of ion transport and photosensory functions. They display several cases of convergent evolution where the same function is present in unrelated or very distant protein groups. Here we report another possible case of such convergent evolution, describing the biophysical properties of a new group of sensory rhodopsins. The first representative of this group was identified in 2004 but none of the members had been expressed and characterized. The well-studied haloarchaeal sensory rhodopsins interacting with methyl-accepting Htr transducers are close relatives of the halobacterial proton pump bacteriorhodopsin. In contrast, the sensory rhodopsins we describe here are relatives of proteobacterial proton pumps, proteorhodopsins, but appear to interact with Htr-like transducers likewise, even though they do not conserve the residues important for the interaction of haloarchaeal sensory rhodopsins with their transducers. The new sensory rhodopsins display many unusual amino acid residues, including those around the retinal chromophore; most strikingly, a tyrosine in place of a carboxyl counterion of the retinal Schiff base on helix C. To characterize their unique sequence motifs, we augment the spectroscopy and biochemistry data by structural modeling of the wild-type and three mutants. Taken together, the experimental data, bioinformatics sequence analyses, and structural modeling suggest that the tyrosine/aspartate complex counterion contributes to a complex water-mediated hydrogen-bonding network that couples the protonated retinal Schiff base to an extracellular carboxylic dyad.

Non-Electroneutrality Generated by Bacteriorhodopsin-Incorporated Membranes Enhances the Conductivity of a Gelatin Memory Device.

We have previously demonstrated the potential of gelatin films as a memory device, offering a novel approach for writing, reading, and erasing through the manipulation of gelatin structure and bound water content. Here, we discovered that incorporating a bacteriorhodopsin (BR)-lipid membrane into the gelatin devices can further increase the electron conductivity of the polypeptide-bound water network and the ON/OFF ratio of the device by two folds. Our photocurrent measurements show that the BR incorporated in the membrane sandwiched in a gelatin device can generate a net proton flow from the counter side to the deposited side of the membrane. This leads to the establishment of non-electroneutrality on the gelatin films adjacent to the BR-incorporated membrane. Our Raman spectroscopy results show that BR proton pumping in the ON state gelatin device increases the bound water presence and promotes polypeptide unwinding compared to devices without BR. These findings suggest that the non-electroneutrality induced by BR proton pumping can increase the extent of polypeptide unwinding within the gelatin matrix, consequently trapping more bound water within the gelatin-bound water network. The resulting rise in hydrogen bonds could expand electron transfer routes, thereby enhancing the electron conductivity of the memory device in the ON state.

Time-resolved investigation of nanometric cell membrane patches with a mid-infrared laser microscope

The proton pump Bacteriorhodopsin (BR) undergoes repeated photocycles including reversible conformational changes upon visible light illumination. Exploiting the sensitivity of infrared (IR) spectra to the conformation, we have determined the reaction kinetic parameters of the conductive intermediate M for the wild-type protein and for its slow mutant D96N during its photocycle. Time-resolved IR micro-spectroscopy using an in-house developed confocal laser microscope operating in the mid-IR is employed to record absorption changes of 10−4 at wavelengths λ1 = 6.08 μm and λ2 = 6.35 μm, assigned to backbone and retinal structural modifications, respectively. Protein samples were embedded in dried lipid bilayers deposited on ultraflat gold supports to enhance the surface field. The signals were analyzed according to a simplified photocycle model with only two dominant states: the dark-adapted state BR* and the intermediate M. We obtained the excitation and relaxation times of the intermediate M from exponential fits to the absorption change time traces. Our results constitute a first step towards future plasmonic-assisted nanoscale time-resolved mid-IR spectrometers for the characterization of bioelectronic and light-harvesting nanodevices based on BR.

Mid-IR quantum cascade laser spectroscopy to resolve lipid dynamics during the photocycle of bacteriorhodopsin.

Membranes are crucial for the functionality of membrane proteins in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interaction-induced dynamics of the protein and the lipid membrane. The photoreceptor and proton pump bacteriorhodopsin (BR) was reconstituted into liposomes, mimicking the native purple membrane. By utilization of deuterated lipid alkyl chains, corresponding vibrational modes are frequency-shifted into a spectrally silent window that allows us to monitor lipid dynamics during the photoreaction of BR. Our home-built quantum cascade laser (QCL)-based IR spectrometer covers all relevant spectral regions to detect both lipid and protein vibrational modes. QCL-probed transients at single wavenumbers are compared with the previously performed step-scan Fourier-transform IR measurements. The absorbance changes of the lipids could be resolved by QCL-measurements with a much better signal-to-noise ratio and with nanosecond time resolution. We found a correlation of the lipid dynamics with the protonation dynamics in the M intermediate. QCL spectroscopy extends the study of the protein's photocycle toward dynamics of the interacting membrane.

Detailed analysis of distorted retinal and its interaction with surrounding residues in the K intermediate of bacteriorhodopsin

The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.

Ion-pumping microbial rhodopsin protein classification by machine learning approach

BackgroundRhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype.ResultsThis study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server (https://bioinfo.imtech.res.in/servers/rhodopred) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers.ConclusionThe overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers.

Placement of Biological Membrane Patches in a Nanofluidic Gap With Control Over Position and Orientation

AbstractPurple membranes from the archaeon Halobacterium salinarum consist of 2D crystals of the light‐driven proton pump bacteriorhodopsin, which convert photons into a proton gradient across the cell membrane. This functional feature and the structural rigidity make them appealing candidates for integration into biomimetic devices. To this end, and in order to carry out their function, purple membranes must be positioned in the correct orientation at the position of interest. Precise placement and control over the orientation of nanoscale objects still constitutes a formidable challenge. Here, it is shown that isolated purple membrane patches can be transported and positioned at predefined locations in nanofluidic confinement, with control over their orientation at the target sites. The transport is achieved through a rocking Brownian motor scheme, while the controlled deposition of the membranes is realized by engineering the surface potential of a fluid‐filled nanofluidic slit. This controlled manipulation of purple membrane patches outlines a new pathway toward the integration of biological or other delicate supramolecular structures into top–down‐fabricated patterns, for the assembly of nanoscale hybrid devices that serve as a light‐driven source of (chemical) energy.

Photoinduced isomerization sampling of retinal in bacteriorhodopsin.

Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, nonvisual light effects, to bacterial light sensing and harvesting. Light-driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here, I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps, hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond together with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins.

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

AbstractTo achieve efficient proton pumping in the light‐driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark‐adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side‐chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground‐state bR.

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Structure/Function Study of Photoreceptive Proteins by FTIR Spectroscopy

Abstract Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive and informative method for studying protein structural changes in photoreceptive proteins. Strong absorption of water in the IR region is always an issue in this method. However, if water content in the sample is controlled during measurements, this method can provide detailed structural information on a single protein-bound water molecule. We optimized the measuring conditions of light-induced difference FTIR spectroscopy to hydrated film samples. In doing so, highly accurate difference FTIR spectra were successfully obtained for a light-driven proton-pump bacteriorhodopsin (BR), not only in the conventional 1800–800 cm−1 region, but also in the 4000–1800 cm−1 region. A highly accurate measuring system of light-induced difference FTIR spectroscopy was applied to various photoreceptive proteins such as animal and microbial rhodopsins, and comprehensive FTIR analyses revealed that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules. It was concluded that a strongly hydrogen-bonded water molecule is the functional determinant of a proton pump. FTIR spectroscopy was also applied to flavin-binding photoreceptors, where we elucidated the molecular mechanisms of adduct formation in the LOV domain, hydrogen-bonding alteration in the BLUF domain, and activation and DNA-repair mechanisms in photolyases. In studies on rhodopsin, we contributed to the discovery and creation of new functions, where FTIR spectroscopy was used for the molecular characterization of new rhodopsins. These new rhodopsins offer promising tools in optogenetics that revolutionized brain sciences. As highlighted in this review article, we provided new insights into the structure/function relationship of biomolecules by unique difference FTIR spectroscopy. In particular, by studying photoreceptive proteins such as rhodopsins, we clarified the mechanism of how light is taken into proteins, and how it leads to their function.

Stationary photocurrent generation from bacteriorhodopsin-loaded lipo-polymersomes in polyelectrolyte multilayer assembly on polyethersulfone membrane.

Vesicles constructed of either synthetic polymers alone (polymersomes) or a combination of polymers and lipids (lipo-polymersomes) demonstrate excellent long-term stability and ability to integrate membrane proteins. Applications using lipo-polymersomes with integrated membrane proteins require suitable supports to maintain protein functionality. Using lipo-polymersomes loaded with the light-driven proton pump bacteriorhodopsin (BR), we demonstrate here how the photocurrent is influenced by a chosen support. In our study, we deposited BR-loaded lipo-polymersomes in a cross-linked polyelectrolyte multilayer assembly either directly physisorbed on gold electrode microchips or cross-linked on an intermediary polyethersulfone (PES) membrane covalently grafted using a hydrogel cushion. In both cases, electrochemical impedance spectroscopic characterization demonstrated successful polyelectrolyte assembly with BR-loaded lipo-polymersomes. Light-induced proton pumping by BR-loaded lipo-polymersomes in the different support constructs was characterized by amperometric recording of the generated photocurrent. Application of the hydrogel/PES membrane support together with the polyelectrolyte assembly decreased the transient current response upon light activation of BR, while enhancing the generated stationary current to over 700nA/cm2. On the other hand, the current response from BR-loaded lipo-polymersomes in a polyelectrolyte assembly without the hydrogel/PES membrane support was primarily a transient peak combined with a low-nanoampere-level stationary photocurrent. Hence, the obtained results demonstrated that by using a hydrogel/PES support it was feasible to monitor continuously light-induced proton flux in biomimetic applications of lipo-polymersomes. Graphical abstract.

Energy Transfer Induced by Dye Encapsulation in a Hybrid Nanoparticle‐Purple Membrane Reversible Assembly

AbstractThe purple membrane (PM) isolated from the bacteria Halobacterium salinarum (H. salinarum) arranges the transmembrane proton pump bacteriorhodopsin (bR) in a 2D hexagonal crystalline lattice. Here, PM sheets containing native bR bend into tube‐like structures with open edges under acidic pH conditions. When decorated with gold nanoparticles (AuNPs), these same PM sheets yield a sealed tube assembly. Upon Rhodamine B (Rh B) sequestration inside the sealed tube, a dramatic decrease in Rh B fluorescence lifetime (τf) from 1.5 ns (unencapsulated) to 14 ps (encapsulated) is observed. The dramatic decrease in lifetime is attributed to energy transfer between AuNPs and Rh B. Subsequent release from the AuNP–PM capsules triggered by an increase in pH shows that 93% of Rh B is recovered (τ = 14 min) due to the capsules' unfolding. The hybrid AuNP–PM material highlights the utility of multicomponent ensembles (i.e., lipid bilayer, protein array, and NPs) by demonstrating that complex, multistimuli 3D responses can lead to multiplexed functions such as controlling energy transfer in the confined, encapsulated state and breaking the energy pair via molecular release in response to a change in pH.

Refilling the proton pump

Protein Dynamics Proteins are dynamic. Rearrangements of side chains, secondary structure, and entire domains gate functional transitions on time scales ranging from picoseconds to milliseconds. Weinert et al. used time-resolved serial crystallography to study large conformational changes in the proton pump bacteriorhodopsin that allow for redistribution of protons during the pumping cycle. They adapted methods used for x-ray free electron lasers to synchrotron x-ray sources. Large loop movements and a chain of water molecules were central to regenerating the starting state of bacteriorhodopsin. Science , this issue p. [61][1] [1]: /lookup/doi/10.1126/science.aaw8634

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography.

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.