Search

The lipopolysaccharides (LPSs) of Rhodobacter are reported to be TLR4 antagonists. Accordingly, the extract of Rhodobacter azotoformans (RAP99) is used as a health supplement for humans and animals in Japan to regulate immune responses in vivo . We previously analyzed the LPS structure of RAP99 (RAP99-LPS) and found it is different from that of E. coli -LPS but similar to lipid A from Rhodobacter sphaeroides (RSLA), a known antagonist of TLR4, with both having three C14 fatty acyl groups, two C10 fatty acyl groups, and two phosphates. Here we show that RAP99-LPS has an immune stimulatory activity and acts as a TLR4 agonist. Pretreatment of RAP99-LPS suppressed E. coli -LPS-mediated weight loss, suggesting it is an antagonist against E. coli -LPS like other LPS isolated from Rhodobacter . However, injections of RAP99-LPS caused splenomegaly and increased immune cell numbers in C57BL/6 mice but not in C3H/HeJ mice, suggesting that RAP99-LPS stimulates immune cells via TLR4. Consistently, RAP99-LPS suppressed the lung metastasis of B16F1 tumor cells and enhanced the expression of TLR3-mediated chemokines. These results suggest that RAP99-LPS is a TLR4 agonist that enhances the activation status of the immune system to promote anti-viral and anti-tumor activity in vivo ., Competing Interests: YH, YN, SF, YT and NT were employed by TFK Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Murakami, Kamimura, Hasebe, Uchida, Abe, Yamamoto, Jiang, Hidaka, Nakanishi, Fujita, Toda, Toda, Tanaka, Akira, Tanaka and Murakami.)

Rhodobacter sp. BT18, a phototrophic salt-resistant bacterium, was isolated from brackish water and screened for the production of exopolysaccharides (EPS). The effect of different light sources on the growth of Rhodobacter sp. BT18 was investigated. The effect on the growth order was found to be blue > white > green > red > yellow > dark. Based on Box-Behnken design, the studied variables (pH 7.0, 35 °C, and 30% of sucrose concentration under 60 h of incubation with blue light illumination) were found to be ideal for the maximum production of EPS (582.5 mg/L). Scanning electron microscopy images revealed the porous nature of EPS. Fourier transform spectroscopy and X-ray diffraction were applied to study the functional groups and the crystalline nature of the EPS, respectively. The emulsification index of the EPS was >75% and the maximum flocculating activity was about 75.4% at 30 mg/L concentration of EPS. In addition, EPS showed effective arsenic (64%) and lead (51%) chelating activities in liquid solutions. The multiple environmental applications of the EPS produced by Rhodobacter sp. BT18 make it be a promising alternative for emulsification, flocculation and metal removal in various industries., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An exopolysaccharide (EPS) producing bacterial strain was isolated from the surface of marine macroalgae (Padina sp.). Based on polyphasic taxonomy, the strain CDR-SL 7Cii was assigned to the genus Rhodobacter and found to be the closest relative of the species Rhodobacter johrii. The strain was able to produce 6.2 g/l of EPS upon fermentation using R2A medium enriched with 2.5% glucose. FT-IR analysis revealed the presence of hydroxyl, carboxyl and diacyl-ester functional groups in the purified EPS. Further Chromatographic study revealed that R. johrii synthesized a high molecular weight anionic exopolysaccharide composed of glucose, glucuronic acid, rhamnose and galactose in a molar ratio of 3:1.5:0.25:0.25. The 1D and 2D NMR spectroscopy (COSY/HSQC) analysis revealed the presence of 1,6 linked-α-d-Glcp, 1,4 linked-β-d-Glcp, 1,3 linked-β-d-GlcA, 1,3 linked-β-d-Galp, 1,6 linked-β-d-Galf and 3-α-l-Rhmp residues. Moreover, the purified EPS has shown stability towards elevated temperature and also acted as a bio-emulsifier to create a high pH and temperature stable emulsion of hydrocarbon/water indicating its industrial potential. This is the first report of EPS production by a strain of Rhodobacter johrii., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Τhe morphology, physiology and immunology, of solid tumors exhibit spatial heterogeneity which complicates our understanding of cancer progression and therapy response. Understanding spatial heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information. We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). We show that endogenous bacteriochlorophyll a in Rhodobacter gives rise to strong optoacoustic signals >800 nm away from interfering endogenous absorbers. Importantly, our results suggest that changes in the spectral signature of Rhodobacter which depend on macrophage activity inside the tumor can be used to reveal heterogeneity of the tumor microenvironment. Employing non-invasive high resolution MSOT in longitudinal studies we show spatiotemporal changes of Rhodobacter spectral profiles in mice bearing 4T1 and CT26.WT tumor models. Accessibility of Rhodobacter to genetic modification and thus to sensory and therapeutic functions suggests potential for a theranostic platform organism.

Lipopolysaccharides (LPS) are components of the Gram-negative bacterial cell surface that stimulate the host innate immune system through the Toll-like receptor (TLR) 4-MD-2 complex. Rhodobacter sp. have been reported to produce LPS that lack endotoxic activity, and instead act as antagonists of other endotoxins. In this report, we focused on LPS, especially the lipooligosaccharide (LOS) fraction produced by Rhodobacter azotoformans that shows production of IL-8, but has an inverse correlation with IL-6 production. We analyzed their molecular structure by using mass spectrometry and nuclear magnetic resonance spectroscopy and report a novel LOS consisting of a shorter glycan structure containing glucuronic acid but not heptoses. A novel glycan structure, Glcα(1 → 4)GlcAα(1 → 4)KDOα(2 → 4)[Glcα(1 → 5)]KDOα(2 → 6)[4-phosphate]GlcNβ(1 → 6) GlcNα1-phosphate, was proposed using NMR methods. The structure was consistent with one obtained based on MS. The MS analysis further revealed the existence of structural variation caused by extension with hexoses. The acyl composition in lipid A was suggested to contain three C14 fatty acyl chains (3-OH-14:0 or 3-oxo-14:0 at N2 of GlcN-1, 3-OH-14:0 at N2 of GlcN-2, that carried another 14:1 Δ7 on its β-hydroxyl group) and two C10 fatty acyl chains (3-OH-10:0 at O3 of both GlcN), which are same as those found in lipid A from Rhodobacter sphaeroides., (Copyright © 2018. Published by Elsevier Ltd.)

An alkali-tolerant, Gram-stain-negative, motile, rod-to-oval-shaped, yellowish brown-colored, phototrophic bacterium, designated as strain JA916 T , was isolated from an alkaline brown pond in Gujarat, India. The DNA G + C content of the strain JA916 T was 65.1 mol%. Strain JA916 T grew well at pH 10. Respiratory quinone was Q-10 and major fatty acid was C 18:1 ω7c/C 18:1 ω6c, with significant quantities of C 15:0 2OH observed. Strain JA916 T shared the highest 16S rRNA gene sequence similarity with the type strains of Rhodobacter johrii (98.4%), followed by Rhodobacter megalophilus (98.3%), Rhodobacter sphaeroides (98.3%), Rhodobacter azotoformans (97.9%) and other members of the genus Rhodobacter (< 97%). 16S rRNA gene-based phylogenetic tree shows that strain JA916 T formed a distinct sub-clade with Rhodobacter johrii, Rhodobacter megalophilus, Rhodobacter sphaeroides and Rhodobacter azotoformans. Further, rpoB-based phylogenetic analysis showed lower similarity with closely related species (≤ 93.0%) of the genus Rhodobacter, which suggests that JA916 T is a novel species of the genus Rhodobacter. DNA-DNA hybridization values between strain JA916 T and related type strains were less than 40%. Phenotypic, chemotaxonomical and phylogenetic differences showed that strain JA916 T was distinct from other species of the genus Rhodobacter, suggesting strain JA916 T represents a new species of the genus for which the name Rhodobacter alkalitolerans sp. nov. is proposed. Type strain is JA916 T (= KCTC 15473 T  = LMG 28749 T ).

The ancient metabolism of photoferrotrophy is likely to have played a key role in the biogeochemical cycle of iron on Early Earth leading to the deposition of Banded Iron Formations prior to the emergence of oxygenic photosynthesis. Extant organisms still performing this metabolism provide a convenient window to peer into its molecular mechanisms. Here we report the molecular structure of FoxE, the putative terminal iron oxidase of Rhodobacter ferrooxidans SW2. This protein is organized as a trimer with two hemes and a disulfide bridge per monomer. The distance between hemes, their solvent exposure and the surface electrostatics ensure a controlled electron transfer rate. They also guarantee segregation between electron capture from ferrous iron and electron release to downstream acceptors, which do not favor the precipitation of ferric iron. Combined with the functional characterization of this protein, the structure reveals how iron oxidation can be performed in the periplasmic space of this Gram-negative bacterium at circumneutral pH, while minimizing the risk of mineral precipitation and cell encrustation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

Several spectrally different types of peripheral light harvesting complexes (LH) have been reported in anoxygenic phototrophic bacteria in response to environmental changes. In this study, two spectral forms of LH2 (T-LH2 and U-LH2) were isolated from Rhodobacter azotoformans. The absorption of T-LH2 was extremely similar to the LH2 isolated from Rhodobacter sphaeroides. U-LH2 showed an extra peak at ∼423 nm in the carotenoid region. To explore the spectral origin of this absorption peak, the difference in pigment compositions of two LH2 was analyzed. Spheroidene and bacteriochlorophyll aP were both contained in the two LH2. And magnesium protoporphyrin IX monomethyl ester (MPE) was only contained in U-LH2. It is known that spheroidene and bacteriochlorophyll aP do not produce ∼423 nm absorption peak either in vivo or in vitro. Whether MPE accumulation was mainly responsible for the formation of the ∼423 nm peak? The interactions between MPE and different proteins were further studied. The results showed that the maximum absorption of MPE was red-shifted from ∼415 nm to ∼423 nm when it was mixed with T-LH2 and its apoproteins, nevertheless, the Qy transitions of the bound bacteriochlorophylls in LH2 were almost unaffected, which indicated that the formation of the ∼423 nm peak was related to MPE-LH2 protein interaction. MPE did not bind to sites involved in the spectral tuning of BChls, but the conformation of integral LH2 was affected by MPE association, the alkaline stability of U-LH2 was lower than T-LH2, and the fluorescence intensity at 860 nm was decreased after MPE combination., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Detergents are widely used for membrane protein research; however, membrane proteins encapsulated in micelles formed by conventional detergents tend to undergo structural degradation, necessitating the development of new agents with enhanced efficacy. Here we prepared several hydrophobic variants of ganglio-tripod amphiphiles (TPAs) derived from previously reported TPAs and evaluated for a multi-subunit, pigment protein superassembly. In this study, TPA-16 was found to be most efficient in protein solubilization while TPA-15 proved most favourable in long-term protein stability. The current study combined with previous TPA studies enabled us to elaborate on a few detergent structure-property relationships that could provide useful guidelines for novel amphiphile design.

A new budding nonsulfur purple bacterium of the genus Rhodobacter (strain Ku-2) was isolated from a mat of a moderately thermal spring (Baikal rift zone, Buryat Republic, Russia). The bacterium had lamellar photosynthetic membranes, which are typica of only one Rhodobacter species, Rba. blasticus. The cells contined spheroidene carotenoids and bacteriochlorophyll a (Bchl a). In vivo absorption spectrum of the cells had the major maximum at 863 nm and an additional peak at 887 nm, which is characteristic of the pigment-protein complexes of Bchl a-containing membranes. The previously described Rba. blasticus strains did not exhibit a 887-nm maximum. The new isolate was photoheterotrophic, with optimal growth occurring at 35 degrees C, 3 g/L NaCl, and pH 7-8. The DNA G+C content was 64.4 mol %. The similarity between the 16S rRNA gene sequences of strain Ku-2 and the Rba. blasticus type strain was 98.7%. The similarity between the PufM amino acid sequences of strain Ku-2 and the previously studied Rba. blasticus strain was 89.0%. Thus, the bacterial strain Ku-2 belonged to the genus Rhodobacter and was phylogenetically related to Rba. blasticus.

Small angle X-ray scattering (SAXS) experiments are performed on two non-ionic surfactants, the dodecyl β-maltoside (DDβM) and the propyl(bi)cyclohexyl α-maltoside (PCCαM), a maltoside derivative containing a rigid bicyclohexyl group as hydrophobic chain, in order to compare the influence of both hydrophobic moiety structure and anomeric form on micelle form factors and intermicellar interactions relevant for membrane protein crystallization. Density and refractive index measurements were performed in order to determine volumetric and optical properties of surfactants, essential for determination of micelle molar masses by both SAXS and SEC-MALLS. SAXS form factors were analyzed by Guinier approximation and inverse Fourier transformation, to obtain the radius of gyration (RG) and the pair distribution function (P(r)) of each surfactant. Form factor model fitting was also performed to describe the shape and the assembly of both surfactant micelles. Finally, second virial coefficients were measured at different percentages of polyethylene glycol 3350, in order to correlate surfactant intermicellar interactions and RC-LH1-PufX phase diagram. It is thus found that while size, shape, and dimensions of micelles are slightly similar for both surfactants, their molar mass and aggregation number differ significantly. PCCαM are more densely packed than DDβM, which reflects (1) an increase in van der Waals contacts between PCCαM hydrophobic chains in the micelle bulk and (2) a supplementary intermicellar attraction compared to DDβM. Finally addition of PEG, which induces a depletion attraction, decreases the solubility of the RC-LH1-PufX complex in PCCαM.

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

In the Rhodobacter (Rba.) species of photosynthetic purple bacteria, a single transmembrane α-helix, PufX, is found within the core complex, an essential photosynthetic macromolecular assembly that performs the absorption and the initial processing of light energy. Despite its structural simplicity, many unresolved questions surround PufX, the most important of which is its location within the photosynthetic core complex. One proposed placement of PufX is at the center of a core complex dimer, where two PufX helices associate in the membrane and form a homodimer. Inability for PufX of certain Rba. species to form a homodimer is thought to lead to monomeric core complexes. In the present study, we employ a combination of computational and experimental techniques to test the hypothesized homodimerization of PufX. We carry out a systematic investigation to measure the dimerization affinity of PufX from four Rba. species, Rba. blasticus , Rba. capsulatus , Rba. sphaeroides , and Rba. veldkampii , using a molecular dynamics-based free-energy method, as well as experimental TOXCAT assays. We found that the four PufX helices have substantially different dimerization affinities. Both computational and experimental techniques demonstrate that species with dimeric core complexes have PufX that can potentially form a homodimer, whereas the one species with monomeric core complexes has a PufX with little to no dimerization propensity. Our analysis of the helix-helix interface revealed a number of positions that may be important for PufX dimerization and the formation of a hydrogen-bond network between these GxxxG-containing helices. Our results suggest that the different oligomerization states of core complexes in various Rba. species can be attributed, among other factors, to the different propensity of its PufX helix to homodimerize.

Nuclear magnetic resonance (NMR) has become a key technology in metabolomics, with the use of stable isotope labeling and advanced heteronuclear multidimensional NMR techniques. In this paper, we focus on the evaluation of extraction solvents to improve NMR-based methodologies for metabolomics. Line broadening is a serious barrier to detecting signals and the annotation of metabolites using multidimensional NMR. We evaluated a series of NMR solvents for easy and versatile single-step extraction using the (13)C-labeled photosynthetic bacterium Rhodobacter sphaeroides, which shows pronounced broadening of NMR signals. The performance of each extraction solvent was judged using 2D (1)H-(13)C heteronuclear single quantum coherence (HSQC) spectra, considering three metrics: (1) distribution of the line width at half height, (2) number of observed signals, and (3) the total observed signal intensity. Considering the total rank values for the three metrics, we chose methanol-d(4) (MeOD) as a semipolar extraction solvent that can sufficiently sharpen the line width and affords better-quality NMR spectra. We also evaluated the series of extraction solvents by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) based ionomics approach. It was also indicated that MeOD is useful for excluding paramagnetic ions as well as macromolecules in an easy single-step extraction. MeOD extraction also appeared to be effective for other bacterial and animal samples. An additional advantage of this semipolar solvent is that it supplements the aqueous (polar) buffer system reported by many groups. The flexible, appropriate application of polar and semipolar extraction should contribute to the large-scale analysis of metabolites.

The aim of this study is to reveal the interaction relationships between lauryl dimethylamine N-oxide (LDAO) and peripheral light-harvesting complex (LH2) as well as the influence of LDAO on structure and function of LH2. In the present work, the effects of LDAO on the conformation and release processes of bacteriochlorophyll (BChl) of LH2 when incubated under different temperature and pH in the presence and absence of LDAO were investigated by spectroscopy. The results indicated that (1) the presence of LDAO resulted in alterations in the conformation, alpha-helix content, and spectra of Tyr and B850 band of LH2 at room temperature and pH 8.0. Moreover, energy transfer efficiency of LH2 was enhanced markedly in the presence of LDAO. (2) At 60 degrees C, both the B800 and B850 band of LH2 were released and transited into free BChl at pH 8.0. However, the release rates of bacteriochlorophylls of B800 and B850 band from LH2 were slowed down and the release processes were changed when incubated in the presence of LDAO. Hence, the stability of LH2 was improved in the presence of LDAO. (3) The accelerated release processes of bacteriochlorophylls of B800 and B850 band of LH2 were induced to transform into bacteriopheophytin (BPhe) and free BChl by LDAO in strong acid and strong alkalic solution at room temperature. However, the kinetic patterns of the B800 and B850 band were remarkably different. The release and self-assemble processes of B850 in LH2 were observed in strong acid solution without LDAO. Therefore, the different release behaviors of B800 and B850 of LH2 are induced by LDAO under different extreme environmental conditions.

Objective: In order to reveal the relationships of compositions and content of pigment in pigment-protein complexes (PPC) and hydrogen photoevolution from anoxygenic phototrophic bacteria., Methods: We isolated and identified pigment-protein complexes using a separation strategy of subsequent fractionated ammonium-sulfate precipitation, ion exchange column chromatography, absorption spectra and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) from hydrogen-producing Rhodobacter azotoformans R7. We investigated the characterizations of the peripheral light-harvesting complex (LH2) with an unusual absorption spectrum by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and fluorescence spectra., Results: We acquired three types of PPC, the reaction center and core light-harvesting complex (RC-LH1) and two kinds of LH2, from strain R7 incubated anaerobically in the light. The two LH2 showed the different absorption spectra, one of them displayed unusual absorption spectrum with the maximum absorption band at 423 nm. The unusual LH2 consisted of two kinds of protein subunits with the molecular weight of 5556.8 Da and 5697.8 Da and carotenoid of spheroidene series with the molecular weight of 562 Da. It was also capable of transferring energy from carotenoid to bacterialcholorophyll and from B800 bacterialcholorophyll to B850 bacterialcholorophyll., Conclusions: Rhodobacter azotoformans R7 with hydrogen-producing capacity could photosynthesize two types of LH2 under anaerobically in the light, one of them presented novel spectral property.

A novel strain, alga-05, of alkaliphilic purple nonsulfur bacteria was isolated from sediments of a small saline (60 g/l) soda lake near Lake Algin (Barguzin Valley, Buryat Republic, Russia). These bacteria contain bacteriochlorophyll a and carotenoids of the alternative spirilloxanthin group with predominating demethylspheroidenone. They are facultative anaerobes; their photosynthetic structures are of the vesicular type and arranged along the cell periphery. Growth of this strain is possible in a salinity range of 5-80 g/l NaCl, with an optimum at 20 g/l NaCl. Best growth occurred at 20-35 degrees C. Analysis of the 16S rRNA gene sequences demonstrated that the studied isolate is closely related to the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis (99% similarity) isolated from soda lakes of the African Rift Zone. According to the results of DNA-DNA hybridization, strain alga-05 has a 52% similarity with the type species of the genus Rhodobaca. On the basis of the obtained genotypic data and some phenotypic properties (dwelling in a hypersaline soda lake of Siberia, moderate halophily, ability to grow at relatively low temperatures, etc.), the isolated strain of purple bacteria was described as a new species of the genus Rhodobaca, Rca. barguzinensis sp. nov.

Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the "classical" phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a near-red (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-A resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IXalpha. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207-212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207-212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.

In the widely studied purple bacterium Rhodobacter sphaeroides, a small transmembrane protein, named PufX, is required for photosynthetic growth and is involved in the supramolecular dimeric organization of the core complex. We performed a structural and functional analysis of the photosynthetic apparatus of Rhodobacter veldkampii, a related species which evolved independently. Time-resolved optical spectroscopy of R. veldkampii chromatophores showed that the reaction center shares with R. sphaeroides spectral and redox properties and interacts with a cytochrome bc(1) complex through a Q-cycle mechanism. Kinetic analysis of flash-induced cytochrome b(561) reduction indicated a fast delivery of the reduced quinol produced by the reaction center to the cytochrome bc(1) complex. A core complex, along with two light-harvesting LH2 complexes significantly different in size, was purified and analyzed by sedimentation, size exclusion chromatography, mass spectroscopy, and electron microscopy. A PufX subunit identified by MALDI-TOF was found to be associated with the core complex. However, as shown by sedimentation and single-particle analysis by electron microscopy, the core complex is monomeric, suggesting that in R. veldkampii, PufX is involved in the photosynthetic growth but is unable to induce the dimerization of the core complex.

The properties of water at the surface, especially at an electrically charged one, differ essentially from those in the bulk phase. Here we survey the traits of surface water as inferred from proton pulse experiments with membrane enzymes. In such experiments, protons that are ejected (or captured) by light-triggered enzymes are traced on their way between the membrane surface and the bulk aqueous phase. In several laboratories it has been shown that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with increase in their electric charge, it was suggested that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. In terms of ordinary electrostatics, the barrier could be ascribed to dielectric saturation of water at a charged surface. In terms of nonlocal electrostatics, the barrier could result from the dielectric overscreening in the surface water layers. It is discussed how the interfacial potential barrier can affect the reactions at interface, especially those coupled with biological energy conversion and membrane transport.

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

The crystal structure of Rhodoferax fermentans high-potential iron protein (HiPIP) has been solved by MAD methods using the anomalous signal from the Fe atoms in the [Fe(4)S(4)] cluster present in the protein and refined to a resolution of 1.45 A. The peptide chain is well defined except in the N- and C-terminal areas. The structure of the protein reveals the presence of three helical fragments, a small beta-sheet and several turns, with the [Fe(4)S(4)] cluster being located close to a surface patch containing several well conserved aromatic residues. The protein fold is very similar to the structures of other known HiPIPs, especially in the region proximal to the [Fe(4)S(4)] cluster, while the largest differences are observed on the opposite side of the protein, which is rich in positive charges and has no sequential homology to other HiPIP families.

Experimental and theoretical results in support of nonlinear dynamic behavior of photosynthetic reaction centers under light-activated conditions are presented. Different conditions of light adaptation allow for preparation of reaction centers in either of two different conformational states. These states were detected both by short actinic flashes and by the switching of the actinic illumination level between different stationary state values. In the second method, the equilibration kinetics of reaction centers isolated from Rhodobacter sphaeroides were shown to be inherently biphasic. The fast and slow equilibration kinetics are shown to correspond to electron transfer (charge separation) at a fixed structure and to combined electron-conformational transitions governed by the bounded diffusion along the potential surface, respectively. The primary donor recovery kinetics after an actinic flash revealed a pronounced dependence on the time interval (deltat) between cessation of a lengthy preillumination of a sample and the actinic flash. A pronounced slow relaxation component with a decay half time of more than 50 s was measured for deltat > 10 s. This component corresponds to charge recombination in reaction centers for which light-induced structural changes have not relaxed completely before the flash. The amplitude of this component depended on the conditions of the sample preparation, specifically on the type of detergent used in the preparation. The redox potential parameters as well as the structural diffusion constants were estimated for samples prepared in different ways.

Positively charged peptide segments of 16 and 18 residues were inserted at a periplasmic turn of the porin from Rhodobacter blasticus in order to form an electric field-dependent plug. The X-ray diffraction analysis of a mutant confirmed that the structure of the porin had remained intact and that the insert was mobile. Incorporation experiments of single molecules into lipid bilayers showed that the distribution of electric conduction increments depended on the field polarity. The observed distributions are explained if the porin molecules enter the bilayer preferentially with their periplasmic surface first. Furthermore, the conduction of membrane-incorporated porin mutants changed reproducibly on field reversal showing asymmetries of reverse similar 15%, while the wild-type remained constant. This asymmetry is most likely caused by the electric field pressing the charged insert onto the pore eyelet in one field direction and removing it from the eyelet in the other. The results encourage attempts to improve the inserts in order to eventually reach diode characteristics.

The absorption (OD) and circular dichroism (CD) spectra of LH2 complexes from various purple bacteria have been measured and modeled. Based on the lineshapes of the spectra we can sort the LH2 complexes into two distinguishable groups: "acidophila"-like (type 1) and "molischianum"-like (type 2). Starting from the known geometric structures of Rhodopseudomonas (Rps.) acidophila and Rhodospirillum (Rsp.) molischianum we can model the OD and CD spectra of all species by just slightly varying some key parameters: the interaction strength, the energy difference of alpha- and beta-bound B850 bacteriochlorophylls (BChls), the orientation of the B800 and B850 BChls, and the (in)homogeneous broadening. Although the ring size can vary, the data are consistent with all the LH2 complexes having basically very similar structures.

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.

The application of electronic structure methods to the prediction of geometries, spin densities, and hyperfine couplings for biologically relevant quinones and semiquinones is reviewed. It is demonstrated that hybrid-type density functional methods are particularly suitable for such studies. Hydrogen bonding to the semiquinone oxygen by appropriate donors is shown to lead to a redistribution of spin density in the carbonyl group of the semiquinone. Experimental trends are well reproduced by the calculated values. Symmetric and asymmetric models of hydrogen bonding are modelled. It is shown that the symmetric models give good agreement with solution studies in vitro. The asymmetric models of hydrogen bonding give quite good agreement with values measured for in vivo semiquinones generated in the reaction centres of the purple photosynthetic bacterium, Rb sphaeroides, and also for the phyllosemiquinone free radical formed during electron transfer in Photosystem I of green plants. These recent advances in electronic structure calculations, in particular the applicability of density functional methods to the study of free radical properties, have opened up an exciting avenue for the complete characterisation of their electronic properties. In particular, the combination of experimental methods of electron paramagnetic resonance and such calculations should in future provide a clearer understanding of free radical chemistry in many areas of biology.

It is becoming increasingly apparent from complete genome sequences that 16S rRNA data, as currently interpreted, does not provide an unambiguous picture of bacterial phylogeny. In contrast, we have found that analysis of insertions and deletions in the amino acid sequences of cytochrome c2 has some advantages in establishing relationships and that this approach may have broad utility in acquiring a better understanding of bacterial relationships. The amino acid sequences of cytochromes c2 and c556 have been determined in whole or in part from four strains of Rhodobacter sulfidophilus. The cytochrome c2 contains three- and eight-residue insertions as well as a single-residue deletion in common with the large cytochromes c2 but in contrast to the small cytochromes c2 and mitochondrial cytochromes. In addition, the Rb. sulfidophilus protein shares a rare six- to seven-residue insertion with other Rhodobacter cytochromes c2. The cytochrome c556 is a low-spin class II cytochrome c homologous to the greater family of cytochromes c', which are usually high-spin. The similarity of cytochrome c556 to other species of class II cytochromes is consistent with the relationships deduced from comparisons of cytochromes c2. Thus, our results do not support placement of Rb. sulfidophilus in a separate genus, Rhodovulum, which was proposed primarily on the basis of 16S rRNA sequences. Instead, the Rhodobacter cytochromes c2 are distinct from those of other genera and species of purple bacteria and show a different pattern of relationships among species than reported for 16S rRNA.

Many of the membrane-bound protein complexes of respiratory and photosynthetic systems are reactive with quinones. To date, no clear structural relationship between sites that bind quinone has been defined, apart from that in the homologous family of "type II" photosynthetic reaction centres. We show here that a structural element containing a weak sequence motif is common to the Q(A) and Q(B) sites of bacterial reaction centres and the Q(i) site of the mitochondrial bc(1) complex. Analyses of sequence databases indicate that this element may also be present in the PsaA/B subunits of photosystem I, in the ND4 and ND5 subunits of complex I and, possibly, in the mitochondrial alternative quinol oxidase. This represents a first step in the structural classification of quinone binding sites., (Copyright 2000 Academic Press.)

The biomass density in biotechnological processes is often determined by indirect and manual methods. Electrical impedance spectroscopy can provide online viable biomass density estimators. In this work, we present two linear estimators obtained with this technique. Four different microorganisms were measured. The detection threshold was approximately 1 g/L (dry weight) for bacteria and 0.5 g/L for yeast. Liposome suspensions were also used to validate the methods. The monitoring of the continuous growth of a yeast culture is also presented.

The genome projects are changing biology by providing the genetic blueprints of entire organisms. The blueprints are tantalizing but we cannot deduce everything we need to know from them, including the structures and detailed functions of proteins. In this paper we describe an approach for obtaining structural information about proteins on a genomic scale. We describe how structural and functional information might eventually be put together to form a basis for describing life at many levels. We then describe how structural information fits into this picture and classes of proteins for which structural information would be useful in a genomic context. We conclude with a proposal for an initiative to determine protein structures on a very large scale.

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185-214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His-tagging. In both cases, a protein product was visible in induced cells. The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography. By N-terminal sequencing of the His-tagged protein, its identity was confirmed.

Photosynthetic organisms fuel their metabolism with light energy and have developed for this purpose an efficient apparatus for harvesting sunlight. The atomic structure of the apparatus, as it evolved in purple bacteria, has been constructed through a combination of x-ray crystallography, electron microscopy, and modeling. The detailed structure and overall architecture reveals a hierarchical aggregate of pigments that utilizes, as shown through femtosecond spectroscopy and quantum physics, elegant and efficient mechanisms for primary light absorption and transfer of electronic excitation toward the photosynthetic reaction center.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

We demonstrate that the B800-830 spectral form of the B800-850 peripheral light-harvesting complex of Rhodobacter sulphidophilus, which is formed at low ionic strengths in the presence of the zwitterionic detergent LDAO, results from a local modification of the bacteriochlorophyll binding site and not the dissociation of the complex. This perturbation does not result in significant changes to the interactions between the pigments as studied by circular dichroism or fluorescence spectroscopy; however, modifications in the pigment binding sites are inferred from changes in the preresonance Raman spectrum. Specifically, an alteration of the hydrogen bonding of the 2-acetyl group of at least one of the bacteriochlorophyll groups that make up the 850 nm absorbing pair is observed. This implies an alteration in the conformation of the C-terminal domain of the alpha-polypeptide, in which are located the two tyrosyl residues that are believed to act as H-bond donors to these groups, induced by the protein-bound detergent in the absence of bound cations. We suggest that the ability of this complex to form an 800-830 complex is linked to the presence of an aspartyl residue immediately upstream of the tyrosyl residues. This study therefore provides a further illustration of the importance of hydrogen bonds to the 2-acetyl group of the bacteriochlorophyll in the determination of its spectral properties; furthermore, we provide a description of a conformational change that is able to modulate chromophore binding in these complexes.

The peripheral light-harvesting bacteriochlorophyll-carotenoid-protein complex B800-850 (LHII) has been isolated from membranes of semi-aerobic dark-grown cells of Rhodobacter sulfidophilus strain W4. A reversed-phase HPLC system resolved one beta- and one alpha-polypeptide in the ratio 1:1. The material obtained was of high purity and suitable for direct microsequence analysis. The primary structures of the beta- and alpha-polypeptides have been determined. The beta-polypeptide consists of 51 amino acid residues, yielding a molecular mass of 5512 Da and having 64.7% hydrophobicity. The alpha-polypeptide consists of 52 amino acid residues, with a calculated molecular mass of 5661 Da and 75% hydrophobicity. The significance of uncommon structure motives with respect to the unusual spectroscopic characteristics of this light-harvesting complex is discussed.

The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three-dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. viridis and Rb. sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

The structure of the membrane channel porin from the phototrophic bacteria Rhodopseudomonas blastica has been refined at 1.96 A resolution yielding an R-factor of 17.6%. The final model consists of all 289 amino acid residues, 247 water molecules and three detergent molecules modelled as n-octyltetraoxyethylene. One of these detergent molecules binds together with its two symmetry-related molecules tightly in a pocket at the molecular 3-fold axis. This pocket may bind three alkyl chains of a lipopolysaccharide which in turn would stabilize the trimer and could possibly play a role in membrane insertion. The overall shape of this porin resembles OmpF of Escherichia coli more than the only known sequence-related porin from Rhodobacter capsulatus. The membrane contacting surface is similar in all structurally known porins; it shows exceptional frequencies of amino acid residues and side-chain rotamers. The 46-residue loop beta 5-beta 6 of the porin is shown to be tightly fastened to the beta-barrel, excluding an in vivo loop movement that closes the pore. The trimer interface region has the structure of a water-soluble protein with an extensive non-polar core and numerous hydrogen bonds at the surface. The loops at the external end of the barrel are long and rigid whereas those at the periplasmic barrel end are short and mobile. The crystal packing is discussed.

By use of computer modelling, we have predicted a model of 16 transmembrane beta-strands for mitochondrial porins structure from human, Saccharomyces cerevisiae, Neurospora crassa and Dictyostelium discoideum. The proposed model takes into account biochemical and immunological data reported in the literature, as well as electrophysiological results obtained with yeast mitochondrial porins with mutations at selected amino acids. The predicted structure is very similar to that of some bacterial porins, as apparent from the homology of their hydropathic profiles.

Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, respectively). The P-->P+ redox transitions could be well separated from redox reactions of other cofactors according to their redox midpoint potential. The IR difference bands of the primary electron donor bacteriochlorophylls all titrate in unison and exhibit the correct midpoint potential. Comparison of the difference spectra with those of isolated bacteriochlorophylls a and b in organic solvents of different polarity and proton activity [Mäntele, W., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472] leads to similar conclusions on the binding and interaction of the pigments within the protein matrix as previously obtained from light-induced P+Q-/PQ difference spectra. Equilibration of the reaction centers in D2O leads to few but distinct shifts of bands and changes of band intensities at 1662, 1634, and 1526 cm-1 (Rhodobacter sphaeroides) and 1694, 1664, 1648, 1630, and 1532 cm-1 (Rhodopseudomonas viridis) as well as to smaller deviations at other wavenumbers. The H-->D-sensitive band at 1662 cm-1 is interpreted in terms of a histidine NH2+ bending mode. A second H/D-sensitive difference band around 1648 cm-1 in the Rhodopseudomonas viridis reaction center may be associated with the peptide C = O of one of the amino acids surrounding P [eventually of the histidine(s) ligating the Mg] which is affected by P+ formation.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of electron transfer between the Rhodobacter sphaeroides R-26 reaction center and nine soluble c-cytochromes have been analyzed and compared to the patterns of the surface electrostatic potentials for each of the proteins. Characteristic first-order electron-transfer rates for 1:1 complexes formed at low ionic strength between the reaction center and the different c-cytochromes were identified and found to vary by a factor of almost 100, while second-order rates were found to differ by greater than 10(6). A correlation was found between the location of likely electrostatic interaction domains on each cytochrome and its characteristic rate of electron transfer. The interaction domains were identified by mapping electrostatic potentials, calculated from the Poisson-Boltzmann equation, onto simulated "encounter surfaces" for each of the cytochromes and the reaction center. For the reaction center, the c-cytochrome binding domain was found to have almost exclusively net negative potential (< -3 kT) and to be shifted slightly toward the M-subunit side of the reaction center. The location of interaction domains of complementary, positive potential (> 3 kT) differed for each cytochrome. The correspondence between electrostatic, structural, and kinetic properties of 1:1 reaction center-cytochrome complexes leads to a proposed mechanism for formation of reaction center-cytochrome electron-transfer complexes that is primarily driven by the juxtaposition of regions of delocalized complementary potential. In this mechanism the clustering of charged residues is of primary importance and not the location of specific residues. A consequence of this mechanism is that many different sets of charge distributions are predicted to be capable of stabilizing a specific configuration for a reaction center-cytochrome complex. This mechanism for reaction center association with water-soluble c-cytochromes fits molecular recognition mechanisms proposed for c-cytochromes in nonphotosynthetic systems. In general, the kinetic scheme for reaction center driven cytochrome oxidation was found to vary between a simple two-state model, involving cytochrome in free and reaction center bound states, and a three-state model, that includes cytochrome binding in kinetically competent ("proximal") and incompetent ("distal") modes. The kinetically incompetent mode of cytochrome binding is suggested not to be an intrinsic feature of the reaction center-cytochrome association but is likely to be due to variation in the physical state of the reaction center.

A method of packing of transmembrane hairpin helices in proteins is described. The procedure is based on the optimization of hydrophobic contacts calculated using the three-dimensional (3D) molecular hydrophobicity potential technique. To verify the validity of the computational scheme, we calculated relative orientations of membrane-spanning peptides in pairs L2-L3, M2-M3, and M4-M5 from L- and M-subunits of the photoreaction center of Rhodopseudomonas viridis and compared the predicted structures with those derived from atomic coordinates. The results of computer modeling agree with the X-ray data. We applied the approach proposed to study possible interhelical interactions in transmembrane hairpin structures of Na+, K(+)-ATPase.

The molecular structure of the high-potential iron-sulfur protein (HiPIP) isolated from the phototrophic bacterium, Rhodocyclus tenuis, has been solved and refined to a nominal resolution of 1.5 A with a crystallographic R-factor of 17.3% for all measured X-ray data from 30 A to 1.5 A. It is the smallest of the HiPIP structures studied thus far with 62 amino acid residues. Crystals used in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A and beta = 90.8 degrees and contained two molecules per asymmetric unit. The structure was solved by a combination of multiple isomorphous replacement with two heavy-atom derivatives, anomalous scattering from the iron-sulfur cluster, symmetry averaging and solvent flattening. The folding motif for this HiPIP is characterized by one small alpha-helix, six Type I turns, an approximate Type II turn and one Type I' turn. As in other HiPIPs, the iron-sulfur cluster is co-ordinated by four cysteinyl ligands and exhibits a cubane-like motif. These cysteinyl ligands are all located in Type I turns. The hydrogen bonding around the metal cluster in the R. tenuis protein is similar to the patterns observed in the Chromatium vinosum and Ectothiorhodospira halophila HiPIPs. Several of the amino acid residues invariant in the previously determined C. vinosum and E. halophila structures are not retained in the R. tenuis molecule. There are 13 solvent molecules structurally conserved between the two R. tenuis HiPIP molecules in the asymmetric unit, some of which are important for stabilizing surface loops. Interestingly, while it is assumed that this HiPIP functions as a monomer in solution, the two molecules in the asymmetric unit pack as a dimer and are related to each other by an approximate twofold rotation axis.