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Retinal-containing light-sensitive proteins – rhodopsins – are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins. [ABSTRACT FROM AUTHOR]

Protein aggregation is the etiopathogenesis of the three most profound vision-threatening eye diseases: age-related cataract, presbyopia, and age-related macular degeneration. This perspective organizes known information on ATP and protein aggregation with a fundamental unrecognized function of ATP. With recognition that maintenance of protein solubility is related to the high intracellular concentration of ATP in cells, tissues, and organs, we hypothesize that (1) ATP serves a critical molecular function for organismal homeostasis of proteins and (2) the hydrotropic feature of ATP prevents pathological protein aggregation while assisting in the maintenance of protein solubility and cellular, tissue, and organismal function. As such, the metabolite ATP plays an extraordinarily important role in the prevention of protein aggregation in the leading causes of vision loss or blindness worldwide. [ABSTRACT FROM AUTHOR]

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid–protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties. [ABSTRACT FROM AUTHOR]

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M−1 cm−1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light–dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process. [ABSTRACT FROM AUTHOR]

Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from Exiguobacterium sibiricum, ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pKa of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes. [ABSTRACT FROM AUTHOR]

Protein aggregation is the etiopathogenesis of the three most profound vision-threatening eye diseases: age-related cataract, presbyopia, and age-related macular degeneration. This perspective organizes known information on ATP and protein aggregation with a fundamental unrecognized function of ATP. With recognition that maintenance of protein solubility is related to the high intracellular concentration of ATP in cells, tissues, and organs, we hypothesize that (1) ATP serves a critical molecular function for organismal homeostasis of proteins and (2) the hydrotropic feature of ATP prevents pathological protein aggregation while assisting in the maintenance of protein solubility and cellular, tissue, and organismal function. As such, the metabolite ATP plays an extraordinarily important role in the prevention of protein aggregation in the leading causes of vision loss or blindness worldwide.

Microbial rhodopsins are the family of retinal-containing proteins that perform primarily the light-driven transmembrane ion transport and sensory functions. They are widely distributed in nature and can be used for optogenetic control of the cellular activities by light. Functioning of microbial rhodopsins results in generation of the transmembrane electric potential in response to a flash that can be measured by direct time-resolved electrometry. This method was developed by L. Drachev and his colleagues at the Belozersky Institute and successfully applied in the functional studies of microbial rhodopsins. First measurements were performed using bacteriorhodopsin from Halobacterium salinarum—the prototype member of the microbial retinal protein family. Later, direct electrometric studies were conducted with proteorhodopsin from Exiguobacterium sibiricum (ESR), the sodium pump from Dokdonia, and other proteins. They allowed detailed characterization of the charge transfer steps during the photocycle of microbial rhodopsins and provided new insights for profound understanding of their mechanism of action. [ABSTRACT FROM AUTHOR]

[Display omitted] • A hydrogen bond regulates dynamics of the O intermediate state of heliorhodopsin. • SNap bond is the key hydrogen bond between transmembrane helices 3 and 4. • A distorted α helix is involved in light-induced conformation changes in heliorhodopsin. • SNap bond exists ubiquitously in heliorhodopsins found in the genome database. Heliorhodopsin (HeR) is a new rhodopsin family discovered in 2018 through functional metagenomic analysis. Similar to microbial rhodopsins, HeR has an all- trans retinal chromophore, and its photoisomerization to the 13- cis form triggers a relatively slow photocycle with sequential intermediate states (K, M, and O intermediates). The O intermediate has a relatively long lifetime and is a putative active state for transferring signals or regulating enzymatic reactions. Although the first discovered HeR, 48C12, was found in bacteria and the second HeR (TaHeR) was found in archaea, their key amino acid residues and molecular architectures have been recognized to be well conserved. Nevertheless, the rise and decay kinetics of the O intermediate are faster in 48C12 than in TaHeR. Here, using a new infrared spectroscopic technique with quantum cascade lasers, we clarified that the hydrogen bond between transmembrane helices (TM) 3 and 4 is essential for the altered O kinetics (Ser112 and Asn138 in 48C12). Interconverting mutants of 48C12 and TaHeR clearly revealed that the hydrogen bond is important for regulating the dynamics of the O intermediate. Overall, our study sheds light on the importance of the hydrogen bond between TM3 and TM4 in heliorhodopsins, similar to the DC gate in channelrhodopsins. [ABSTRACT FROM AUTHOR]

The first member and eponym of the rhodopsin family was identified in the 1930s as the visual pigment of the rod photoreceptor cell in the animal retina. It was found to be a membrane protein, owing its photosensitivity to the presence of a covalently bound chromophoric group. This group, derived from vitamin A, was appropriately dubbed retinal. In the 1970s a microbial counterpart of this species was discovered in an archaeon, being a membrane protein also harbouring retinal as a chromophore, and named bacteriorhodopsin. Since their discovery a photogenic panorama unfolded, where up to date new members and subspecies with a variety of light-driven functionality have been added to this family. The animal branch, meanwhile categorized as type-2 rhodopsins, turned out to form a large subclass in the superfamily of G protein-coupled receptors and are essential to multiple elements of light-dependent animal sensory physiology. The microbial branch, the type-1 rhodopsins, largely function as light-driven ion pumps or channels, but also contain sensory-active and enzyme-sustaining subspecies. In this review we will follow the development of this exciting membrane protein panorama in a representative number of highlights and will present a prospect of their extraordinary future potential.

[Display omitted] • The review article provides an overview of the understanding of the molecular properties of Opn5 and its homologs. It outlines possible strategies for manipulation of animal opsins related to Opn5, and their potential optogenetic applications of these opsins. • Members of the Opn5 group found in vertebrate genomes can be classified into phylogenetically distinct subgroups: Opn5m, Opn5m2, Opn5L1, Opn5L2, and Opn6. Invertebrates possess Opn5 homologs that are independent of the vertebrate Opn5 subgroups. • Opn5m, Opn5m2, and Opn5L2 are UV-sensitive bistable opsins. Opn5L1 is active in its dark state and is inactivated upon visible light absorption. • The amino acid at position 188 is a key residue for the molecular properties of both Opn5m and Opn5L1. Mutating this residue can change the molecular properties of Opn5m, Opn5L1, and vertebrate rhodopsin. • Opn5m and Opn5L1 are potential optogenetic tools as a UV-sensitive Gq-coupled GPCR and a photo-inactivatable constitutively active GPCR, respectively." Animal opsin is a G-protein coupled receptor (GPCR) and binds retinal as a chromophore to form a photopigment. The Opsin 5 (Opn5) group within the animal opsin family comprises a diverse array of related proteins, such as Opn5m, a protein conserved across all vertebrate lineages including mammals, and other members like Opn5L1 and Opn5L2 found in non-mammalian vertebrate genomes, and Opn6 found in non-therian vertebrate genomes, along with Opn5 homologs present in invertebrates. Although these proteins collectively constitute a single clade within the molecular phylogenetic tree of animal opsins, they exhibit markedly distinct molecular characteristics in areas such as retinal binding properties, photoreaction, and G-protein coupling specificity. Based on their molecular features, they are believed to play a significant role in physiological functions. However, our understanding of their precise physiological functions and molecular characteristics is still developing and only partially realized. Furthermore, their unique molecular characteristics of Opn5-related proteins suggest a high potential for their use as optogenetic tools through more specialized manipulations. This review intends to encapsulate our current understanding of Opn5, discuss potential manipulations of its molecular attributes, and delve into its prospective utility in the burgeoning field of animal opsin optogenetics. [ABSTRACT FROM AUTHOR]

Resonance energy transfer in self-assembled hybrid structures formed by water-soluble semiconductor CdSe/ZnS quantum dots (QD) with a cysteine shell and the Exiguobacterium sibiricum retinal protein (ESR) in three modifications, with and without a 6-membered C-terminal histidine tag, and with both the histidine tag and C-terminal cysteine residue. Steady-state and time-resolved fluorescence spectroscopy was used to demonstrate that nonradiative energy transfer from QD onto the ESR protein depends on the strength of intermolecular interactions in the hybrid complex. A shift from electrostatic interactions in the QD-ESR donor-acceptor pair to coordinate bond with an additional disulfide bond resulted in an increase of the energy transfer efficiency from 40 to over 90%. The proposed method to produce noncovalent conjugates from QDs and light-sensitive proteins can be a promising one for biomedical applications, as well as for the development of new solar energy accumulation systems. [ABSTRACT FROM AUTHOR]

A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5 ms and 4.5 ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH 8.4, a spectrally silent electrogenic component with τ 0.25 ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH 5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer. [ABSTRACT FROM AUTHOR]

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggered trans- cis isomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I in Natronomonas pharaonis halorhodopsin ( NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals of Halobacterium salinarum halorhodopsin ( HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast to NpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found in NpHR. Firstly, the partially chloride-depleted form of HsHR has increased normalized B factors in the region of helix C that is close to CBS I and changes its conformation in NpHR. Secondly, prolonged soaking of HsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form of HsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport. [ABSTRACT FROM AUTHOR]

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's p K to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins. [ABSTRACT FROM AUTHOR]

Halorhodopsin from the halophilic archaeon Halobacterium salinarum is a membrane located light-driven chloride pump. Upon illumination Halorhodopsin undergoes a reversible photocycle initiated by the all- trans to 13- cis isomerization of the covalently bound retinal chromophore. The photocycle consists of several spectroscopically distinct intermediates. The structural basis of the chloride transport mechanism remains elusive, presumably because packing contacts have so far precluded protein conformational changes in the available crystals. With the intention to structurally characterize late photocycle intermediates by X-ray crystallography we crystallized Halorhodopsin in a new crystal form using the vesicle fusion method. In the new crystal form lateral contacts are mediated by helices A and G. Helices E and F that were suggested to perform large movements during the photocycle are almost unrestrained by packing contacts. This feature might permit the displacement of these helices without disrupting the crystal lattice. Therefore, this new crystal form might be an excellent system for the structural characterization of late Halorhodopsin photocycle intermediates by trapping or by time resolved experiments, especially at XFELs. [ABSTRACT FROM AUTHOR]

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum , ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively. • ESR is a proton pump with an unusual H+ donor to the Schiff base (SB), Lys96. • Asp and Glu effectively replace Lys as H+ donors in the K96D/A47T and K96E mutants. • Reprotonation of the SB is pH-dependent in ESR and its mutants. • The donors in ESR connect to the SB and the bulk in the M → N transition. • The carboxylic H+ donors are reprotonated quickly after transferring H+ to SB in ESR. [ABSTRACT FROM AUTHOR]

Abstract: Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks. [Copyright &y& Elsevier]

The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all- trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13- cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101aR2, Asp96bR) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This implies that the structural property of O1 in the aR2 photocycle can be investigated over a wide pH range. [Display omitted] • Two substates (O1 and O2) with distinct spectral properties occur in the O intermediate of aR2. • O1 (580 nm) occurs as a transition state between N (540 nm) and O2 (605 nm). [ABSTRACT FROM AUTHOR]

ABSTRACT Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light-driven proton pump. Here, we report the three-dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 ( a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein-protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C-terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] The structure of the D75N mutant of sensory rhodopsin II in complex with its cognate transducer is presented. [•] The topology of the complex is “U”-shape. [•] The changes in the hydrogen bonds network are observed. [•] The implications on the prospective mechanism of functioning are discussed. [Copyright &y& Elsevier]

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λmax (540 nm) of the mutant. A long-lived substate of O with a red-shifted λmax (∼565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (Oslow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In Oslow, however, the main chain of Lys216 is less distorted and retinal takes on the 13- cis/15- syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the p Ka value of which is suggested to be much lower in Oslow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary: The hexagonal P622 crystal of bacteriorhodopsin, which is made up of stacked membranes, is stable provided that the precipitant concentration in the soaking solution is higher than a critical value (i.e., 1.5 M ammonium sulfate). Diffraction data showed that the crystal lattice shrank linearly with increasing precipitant concentration, due primarily to narrowing of intermembrane spaces. Although the crystal shrinkage did not affect the rate of formation of the photoreaction M intermediate, its lifetime increased exponentially with the precipitant concentration. It was suggested that the energetic barrier of the M-to-N transition becomes higher when the motional freedom of the EF loop is reduced by crystal lattice force. As a result of this property, the M state accumulated predominantly when the crystal that was soaked at a high precipitant concentration was illuminated at room temperature. Structural data obtained at various pH levels showed that the overall structure of M is not strongly dependent on pH, except that Glu194 and Glu204 in the proton release complex are more separated at pH 7 than at pH 4.4. This result suggests that light-induced disruption of the paired structure of Glu194 and Glu204 is incomplete when external pH is lower than the pK a value of the proton release group in the M state. [Copyright &y& Elsevier]

Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of ≈90. Multidimensional spectroscopy of U-13C,15N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR568 and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP. [ABSTRACT FROM AUTHOR]

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-Å resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the PK[suba] of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as ≈45%, and the 46° angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer. [ABSTRACT FROM AUTHOR]

Abstract: Xanthorhodopsin is a light-driven proton pump like bacteriorhodopsin, but made more effective for collecting light by its second chromophore, salinixanthin, a carotenoid. Action spectra for transport and fluorescence of the retinal upon excitation of the carotenoid indicate that the carotenoid functions as an antenna to the retinal. The calculated center-to-center distance and angle of the transition moments of the two chromophores are 11 Å and 56°, respectively. As expected from their proximity, the carotenoid and the retinal closely interact: tight binding of the carotenoid, as indicated by its sharpened vibration bands and intense induced circular dichroism in the visible, is removed by hydrolysis of the retinal Schiff base, and restored upon reconstitution with retinal. This antenna system, simpler than photosynthetic complexes, is well-suited to study features of excited-state energy migration. [Copyright &y& Elsevier]

By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The 15N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult as judged by the multiple I substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photo- cycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer. [ABSTRACT FROM AUTHOR]