List Of Publications

Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. A mechanism that exists in some cyanobacterial species, enables non-photochemical quenching of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP). The OCP contains an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (CTDH) and the NTD (referred to as helical carotenoid proteins, HCP). The CTDH facilitates uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structure of the holo-HCP4 binding canthaxanthin molecule and an improved structure of the apo-CTDH from Anabaena sp. PCC 7120. We propose here models of the binding of the HCP4 to the PBS and the associated energy quenching mechanism. Our results show that the presence of the carotenoid is essential for fluorescence quenching. We also examined interactions within OCP-like species, including HCP4 and CTDH, providing the basis for mechanisms of carotenoid transfer from CTDH to HCPs.

Enzymes have gained their unique efficiency and catalytic activity through billions of years of evolution, perfecting their active site to a desired reaction. Inspired by nature, a novel enzyme-mimicking platform is designed based on stable protein 1 (SP1) to create a nano-compartment that mimics peroxidase activity. The biohybrid reveals enhanced activity over the hemin cofactor alone and improved stability in organic solvents in comparison to native peroxidase. Furthermore, the utilization of the obtained biohybrid in an optical glucose biosensing platform is shown. The biohybrid crystallographic structure is solved, indicating that the SP1 structure is not affected by the hemin coordination. This work opens the path for developing new cofactor binding centers in engineered protein scaffolds for various artificial catalytic processes..

Photosynthetic organisms adapt to changing light conditions by manipulating their light harvesting complexes. Biophysical, biochemical, physiological and genetic aspects of these processes are studied extensively. The structural basis for these studies is lacking. In this study we address this gap in knowledge by focusing on phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. In this study we focus on the phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. Specifically, we examine red algae (Porphyridium purpureum) grown under a low light intensity (LL) and a medium light intensity (ML). Using cryo-electron microscopy, we resolve the structure of ML-PBS and compare it to the LL-PBS structure. The ML-PBS is 13.6 MDa, while the LL-PBS is larger (14.7 MDa). The LL-PBS structure have a higher number of closely coupled chromophore pairs, potentially the source of the red shifted fluorescence emission from LL-PBS. Interestingly, these differences do not significantly affect fluorescence kinetics parameters. This indicates that PBS systems can maintain similar fluorescence quantum yields despite an increase in LL-PBS chromophore numbers. These findings provide a structural basis to the processes by which photosynthetic organisms adapt to changing light conditions.

2-Hydroxybiphenyl 3-monooxygenase (HbpA) is an FAD dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. We have determined the structure of HbpA from the soil bacterium Pseudomonas azelaica HBP1 with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5Å to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. The entrance to the active site was confirmed by generating variant G255F which exhibited only 7% of the wild-type’s specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

Feingold syndrome 1 (FGLDS1) is an autosomal dominant malformation syndrome, characterized by skeletal anomalies, microcephaly, facial dysmorphism, gastrointestinal atresias and learning disabilities. Mutations in the MYCN gene are known to be the cause of this syndrome. Congenital absence of the flexor pollicis longus (CAFPL) tendon is a rare hand anomaly. Most cases are sporadic and no genetic variants have been described associated with this abnormality. We describe here a pedigree combining familial CAFPL tendon as a feature of FGLDS1. Molecular analyses of whole exome sequence data in five affected family members spanning three generations of this family revealed a novel mutation in the MYCN gene (c.1171C>T; p.Arg391Cys). Variants in MYCN have not been published in association with isolated or syndromic CAFPL tendon, nor has this been described as a skeletal feature of Feingold syndrome. This report expands on the clinical and molecular spectrum of MYCN-related disorders and highlights the importance of MYCN protein in normal human thumb and foramen development.

The enzyme 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys11 was replaced by Asn (ApC11N), and in the metal-independent Escherichia coliKDO8P synthase a reciprocal mutation, Asn26 to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (kcat/Km) was reduced by only ∼12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn2+ or Cd2+ stimulated its activity to ∼30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.

Chemistry courses in higher education have traditionally been composed of lectures, problem solving sessions, and laboratories. This study describes a Web-based chemistry course and the learning outcomes of freshmen that used it. Chemistry faculty and teaching assistants were interviewed regarding their views about Web-based teaching and learning. Students who took part in a Web-based general chemistry course were divided into two groups based on their preference of participating in a Computerized Molecular Modeling (CMM) project. The experimental group students carried out an individualized project using CMM software to represent a complex molecule in three model types, compute its molecular weight, and construct hybridization and electrical charge distribution for each of the carbon atoms in the molecule. Pre- and post-tests along with final examination grades served for assessing the students’ achievements. The 95 experimental students achieved significantly higher grades than their 120 control-group peers in both the post-test and the final examination. The experimental students were able to switch from 1-D to 2- and 3-D molecular representations, argue for selecting an appropriate substance for a particular purpose, and transfer between the four levels of understanding in chemistry better than their control counterparts.

We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm−1 is assigned to the C–C stretching vibration while the mode at 454 cm−1 is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm−1 does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm−1 rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N–H rocking in-plan vibration.

X-ray crystal structures of the isolated phycobiliprotein components of the phycobilisome have provided high resolution details to the description of this light harvesting complex at different levels of complexity and detail. The linker-independent assembly of trimers into hexamers in crystal lattices of previously determined structures has been observed in almost all of the phycocyanin (PC) and allophycocyanin (APC) structures available in the Protein Data Bank. In this paper we describe the X-ray crystal structures of PC and APC from Synechococcus elongatus sp. PCC 7942, PC from Synechocystis sp. PCC 6803 and PC from Thermosynechococcus vulcanus crystallized in the presence of urea. All five structures are highly similar to other PC and APC structures on the levels of subunits, monomers and trimers. The Synechococcus APC forms a unique loose hexamer that may show the structural requirements for core assembly and rod attachment. While the Synechococcus PC assembles into the canonical hexamer, it does not further assemble into rods. Unlike most PC structures, the Synechocystis PC fails to form hexamers. Addition of low concentrations of urea to T. vulcanus PC inhibits this proteins propensity to form hexamers, resulting in a crystal lattice composed of trimers. The molecular source of these differences in assembly and their relevance to the phycobilisome structure is discussed.

Allophycocyanin (APC) is the primary pigment–protein component of the cores of the phycobilisome antenna complex. In addition to an extremely high degree of amino acid sequence conservation, the overall structures of APC from both mesophilic and thermophilic species are almost identical at all levels of assembly, yet APC from thermophilic organisms should have structural attributes that prevent thermally induced denaturation. We determined the structure of APC from the thermophilic cyanobacterium Thermosynechococcus vulcanus to 2.9 Å, reaffirming the conservation of structural similarity with APC from mesophiles. We provide spectroscopic evidence that T. vulcanus APC is indeed more stable at elevated temperatures in vitro, when compared with the APC from mesophilic species. APC thermal and chemical stability levels are further enhanced when monitored in the presence of high concentrations of buffered phosphate, which increases the strength of hydrophobic interactions, and may mimic the effect of cytosolic crowding. Absorption spectroscopy, size-exclusion HPLC, and native gel electrophoresis also show that the thermally or chemically induced changes in the APC absorption spectra that result in the loss of the prominent 652-nm band in trimeric APC are not a result of physical monomerization. We propose that the bathochromic shift that occurs in APC upon trimerization is due to the coupling of the hydrophobicity of the α84 phycocyanobilin cofactor environment created by a deep cleft formed by the β subunit with highly charged flanking regions. This arrangement also provides the additional stability required by thermophiles at elevated temperatures. The chemical environment that induces the bathochromic shift in APC trimers is different from the source of shifts in the absorption of monomers of the terminal energy acceptors APCB and LCM, as visualized by the building of molecular models.

Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction.

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

We have recently reported the crystallization of the reaction center of Photosystem II in the presence of detergent mixtures [Adir N (1999) Acta Crystallogr D Biol Crystallogr D55: 891–894]. We have used high performance liquid chromatography, dynamic light scattering, native gel electrophoresis and thermoluminescence measurements to characterize the interaction between these detergent mixtures and RC II, to try and understand their role in the crystallization process. Size exclusion HPLC and dynamic light scattering confirmed that the isolated RC II used for crystallization was exclusively monomeric. Dynamic light scattering measurements show that the detergent mixtures formed single micelles within a limited range of hydrodynamic radii. Both size exclusion HPLC and dynamic light scattering were used to follow the interaction between the detergent mixtures and monomeric RC II. These techniques revealed a decrease in the detergent mixture treated RC II particle size (with respect with the untreated RC II), and that RC II from solubilized crystals contained particles of the same size. Native gel electrophoresis showed that this change in apparent size is not due to the disintegration of the internal structure of the RC II complex. Thermoluminescence measurements of solubilized RC II crystals showed charge recombination from the S2,3QA −state, indicating that RC II remains functionally viable following detergent mixture treatment and crystallization. The role of the detergent mixtures in the crystallization of RC II is discussed.

We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2 · 10−3) was detected along the c-axis. Under heat treatment above 200 °C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.

The cyanobacterium Synechocystis sp. PCC 6803 imports Mn2+ ions via MntCAB, an ABC transport system that is expressed at submicromolar Mn2+ concentrations. The structures of the wild type (WT) and a site-directed mutant of the MntC solute-binding protein have been determined at 2.7 and 3.5 Å resolution, respectively. The WT structure is significantly improved over the previously determined structure (PDB entry 1xvl ), showing improved Mn2+ binding site parameters, disulfide bonds in all three monomers and ions bound to the protein surface, revealing the role of Zn2+ ions in the crystallization liquor. The structure of MntC reveals that the active site is surrounded by neutral-to-­positive electrostatic potential and is dominated by a network of polar interactions centred around Arg116. The mutation of this residue to alanine was shown to destabilize loops in the entrance to the metal-ion binding site and suggests a possible role in MntC function.

The process of photosynthesis is initiated by the absorption of light energy by large arrays of pigmentsbound in an ordered fashion within protein complexes called antennas. These antennas transfer the absorbedenergy at almost 100% efficiency to the reaction centers that perform the photochemical electron transferreactions required for the conversion of the light energy into useful and storable chemical energy. Inprokaryotic cyanobacteria, eukaryotic red algae and cyanelles, the major antenna complex is called the phycobilisome,an extremely large (3–7 MDa) multi subunit complex found on the stromal side of the thylakoidmembrane. Phycobilisomes are assembled in an ordered sequence from similarly structured units that covalentlybind a variety of linear tetrapyrolle pigments called bilins. Phycobilisomes have a broad cross-sectionof absorption (500–680 nm) and mainly transfer the absorbed energy to photosystem II. Theycan, however, function as an antenna of photosystem I, and their composition can be altered as a resultof changes in the environmental light quality. The phycobilisome is structurally and functionally differentfrom other classes of photosynthetic antenna complexes. In this review, we will describe the importantstructural and functional characteristics of the phycobilisome complex and its components, especially withrespect to its assembly and disassembly.

Autosomal recessive congenital ichthyosis (ARCI) refers to a large and genetically heterogenous group of non-syndromic disorders of cornification featuring diffuse scaling. Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome is a rare autosomal recessive syndromic form of ichthyosis. The disease usually results from premature termination codon-causing pathogenic variants in CLDN1encoding CLAUDIN-1 (CLDN1). We used whole exome sequencing (WES), Sanger sequencing, 3D protein modeling, Western blotting, and immunofluorescence confocal microscopy to delineate the genetic basis of ichthyosis in two siblings with ichthyosis but no other ectodermal abnormalities. One of the two siblings underwent liver transplantation in early childhood due to biliary atresia. Both patients were found to carry a homozygous missense pathogenic variant, c.242G>A (p.Arg81His), in CLDN1. The variant resulted in decreased CLDN1 expression in patient skin. 3D protein modeling predicted that p.Arg81His induces deleterious conformational changes. Accordingly, HaCaT cells transfected with a construct expressing the mutant CLDN1 cDNA featured decreased levels and mislocation of CLDN1 as compared with cells expressing the wildtype cDNA. In conclusion, we describe the first pathogenic missense variant in CLDN1shown to result in ARCI.

The conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Bio-photoelectrochemical cells (BPECs) utilizing unicellular photosynthetic microorganisms have been studied, however similar harvesting of electrons from more evolved intact photosynthetic organisms has not been previously reported. In this study, we describe for the first time BPECs containing intact live marine macroalgae (seaweeds) in natural seawater or saline buffer. The BPECs produce electrical currents of >50 mA/cm2, from both light-dependent (photosynthesis) and light-independent processes. These values are significantly greater than the current densities that have been reported for single-cell microorganisms. The photocurrent is inhibited by the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating that the source of light-driven electrons is from photosynthetic water oxidation. The current is mediated to the external anode via NADPH and possibly other reduced molecules. We show that intact macroalgae cultures can be used in large-scale BPECs containing seawater, to produce bias-free photocurrents, paving the way for the future development of low-cost energy solar energy conversion technologies using BPECs.

Small heat shock proteins (sHSPs) belong to the superfamily of molecular chaperones. They prevent aggregation of partially unfolded or misfolded client proteins, providing protection to organisms under stress conditions. Here, we report the biophysical and structural characterization of a small heat shock protein (HspA) from a thermophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 M urea. HspA has been shown to be important for the protection of Photosystem II and the Phycobilisomeantenna complex at elevated temperatures. Heterologously expressed HspA requires the presence of 1–2 M urea to maintain its solubility at concentrations required for most characterization methods. Spectroscopic studies reveal the presence of the β-sheet structure and intactness of the tertiary fold in HspA. In vitro assays show that the HspA maintains chaperone-like activity in protecting soluble proteins from thermal aggregation. Chromatography and electron microscopy show that the HspA exists as a mixture of oligomeric forms in the presence of 2 M urea. HspA was successfully crystallized only in the presence of 2 M urea. The crystal structure of HspA shows urea-induced loss of about 30% of the secondary structure without major alteration in the tertiary structure of the protein. The electron density maps reveal changes in the hydrogen bonding network which we attribute to the presence of urea. The crystal structure of HspA demonstrates a mixture of both direct interactions between urea and protein functionalities and interactions between urea and the surrounding solvent that indirectly affect the protein, which are in accordance with previously published studies.

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient’s skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations.

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF4] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates L-tyrosine and L-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.

Autosomal recessive congenital ichthyosis (ARCI) refers to a large and genetically heterogenous group of non-syndromic disorders of cornification featuring diffuse scaling. Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome is a rare autosomal recessive syndromic form of ichthyosis. The disease usually results from premature termination codon-causing pathogenic variants in CLDN1encoding CLAUDIN-1 (CLDN1). We used whole exome sequencing (WES), Sanger sequencing, 3D protein modeling, Western blotting, and immunofluorescence confocal microscopy to delineate the genetic basis of ichthyosis in two siblings with ichthyosis but no other ectodermal abnormalities. One of the two siblings underwent liver transplantation in early childhood due to biliary atresia. Both patients were found to carry a homozygous missense pathogenic variant, c.242G>A (p.Arg81His), in CLDN1. The variant resulted in decreased CLDN1 expression in patient skin. 3D protein modeling predicted that p.Arg81His induces deleterious conformational changes. Accordingly, HaCaT cells transfected with a construct expressing the mutant CLDN1 cDNA featured decreased levels and mislocation of CLDN1 as compared with cells expressing the wildtype cDNA. In conclusion, we describe the first pathogenic missense variant in CLDN1shown to result in ARCI.

Translin is a human octameric protein that specifically binds the single-stranded microsatellite repeats d(GT)n and the corresponding transcripts (GU)n. It also binds, with lesser affinities, other single-stranded G-rich DNA and RNA sequences. TRAX is a human protein that bears a homology to Translin and interacts with it. Translin and TRAX have been proposed to be involved in DNA recombination, chromosomal translocation and mRNA transport and translation. Both proteins are highly conserved in eukaryotes, including the fission yeast Schizosaccharomyces pombe, which is amenable to genetic analysis. Here, we report the first study of the S.pombe Translin and TRAX homologs. We have deleted the genes encoding Translin and TRAX in S.pombe and found that the proliferation of the mutant cells was slightly stimulated, suggesting that these genes are not essential for the fission yeast. We have also shown that the S.pombe Translin and TRAX interact. Biochemical analysis of the S.pombeTranslin, which was cloned and expressed in Escherichia coli, revealed that it is octameric and that it selectively binds d(GT)n and d(GTT)n microsatellite repeats. However, unlike the human protein, it has much higher affinities for the homologous RNA sequences (GU)n and (GUU)n. These data suggest that the S.pombe Translin is primarily involved in functions related to RNA metabolism.

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS−PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 Å. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 Å. The crystals belong to the tetragonal space group P43212, with unit cell dimensions of a = b = 142.7 Å and c = 254.8 Å. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC−cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 ± 0.1 μs in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC−cyt c2 complex:  (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of the RC−cyt c2 complex.

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.

Cyanobacteria light-harvesting complexes can change their structure to cope with fluctuating environmental conditions. Studying in vivo structural changes is difficult owing to complexities imposed by the cellular environment. Mimicking this system in vitro is challenging, as well. The in vivo system is highly concentrated, and handling similar in vitro concentrated samples optically is difficult because of high absorption. In this research, we mapped the cyanobacteria antennas self-assembly pathways using highly concentrated solutions of phycocyanin (PC) that mimic the in vivo condition. PC was isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus and measured by several methods. PC has three oligomeric states: hexamer, trimer, and monomer. We showed that the oligomeric state was changed upon increase of PC solution concentration. This oligomerization mechanism may enable photosynthetic organisms to adapt their light-harvesting system to a wide range of environmental conditions.

Understanding of electronic energy transition (EET) mechanisms from the light-harvesting unit to the reaction center in a natural system has been limited by (a) the use of conventional transient time-resolved spectroscopy at room temperature, which result in high signal-to-noise ratio and (b) examining extracts instead of intact light-harvesting units. Here, we report previously unknown differences and new insight in EET of two cyanobacteria species, Acaryochloris marina and Thermosynechoccocus vulcanus, which have been found only after using UV–vis, hole-burning, and fluorescence spectroscopy at ultralow temperature and examining their intact light-harvesting unit, phycobilisomes (PBS). Although the exciton formation is similarly induced by photoexcitation of chromophore assemblies in phycocyanin (PC) and allophycocyanin (APC) in PBSs of both species, the EET mechanisms are totally different, being adiabatic in A. marina and nonadiabatic in T. vulcanus. The PBS of A. marina has only one APC trimer and energy transfer is through coupling of α84 in APC with β84 in adjacent PC. In T. vulcanus, the PBS has three components: coupling between APC core and the entire PC rod and couplings of β–β18 and of LCM to β in the adjacent APC-like trimer. A total of 80% of the excitation energy is trapped in the coupling β–β18 and regulates the flow of energy from the high- to low-level terminal electronic transition emitter β-LCM. All these details cannot be observed at room temperature and in extracted units.

The enzyme 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS) catalyses the condensation of arabinose 5-phosphate (A5P) and phosphoenol pyruvate (PEP) to obtain 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P). We have elucidated initial modes of ligand binding in KDO8PS binary complexes by X-ray crystallography. Structures of the apo-enzyme and of binary complexes with the substrate PEP, the product KDO8P and the catalytically inactive 1-deoxy analog of arabinose 5-phosphate (1dA5P) were obtained. The KDO8PS active site resembles an irregular funnel with positive electrostatic potential situated at the bottom of the PEP-binding sub-site, which is the primary attractive force towards negatively charged phosphate moieties of all ligands. The structures of the ligand-free apo-KDO8PS and the binary complex with the product KDO8P visualize for the first time the role of His202 as an active-site gate. Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P shows these ligands bound to the enzyme in the PEP-binding sub-site, and not as expected to the A5P sub-site. Taken together, the structures presented here strengthen earlier evidence that this enzyme functions predominantly through positional catalysis, map out the roles of active-site residues and provide evidence that explains the total lack of catalytic reversibility.

The crystal structures of 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (Kd = 0.4 μM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 Å resolution, respectively. Both the KDOPS·PEP and KDOPS·inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 Å, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Cα atoms indicates an rms difference of 0.41 Å. The PEP in the KDOPS·PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS’s selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P−O−C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and d-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-d-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.

Tyrosinases are type 3 copper enzymes that are involved in the production of melanin and have two copper ions in the active site. Here, the crystallization and primary analysis of a tyrosinase from Bacillus megaterium is reported. The purified protein was crystallized in the absence or presence of zinc ions and the crystals diffracted to a resolution of 2.0 Å. Crystals obtained in the presence of zinc belonged to space group P212121, while crystals grown in the absence of zinc belonged to space group P21. In both space groups the asymmetric unit contained a dimer, with minor differences in the crystal density and in packing interactions.

The manganese-stabilizing protein (MSP) of Photosystem II was purified from spinach photosynthetic membranes. The MSP was crystallized in the presence of calcium. Despite the apparent purity of the isolated protein, the crystals grew to only about 0.05 mm in their largest dimension. The MSP was analyzed to identify possible sources of protein heterogeneity that could hinder crystal growth. Tandem reverse-phase HPLC/ electronspray ionization mass spectrometry analysis of the MSP showed a major peak and four smaller peaks. All five peaks had molecular masses of 26 535, as expected for mature MSP, indicating the absence of heterogeneities due to covalent modifications. MALDI mass spectroscopy was utilized to identify heterogeneities in the MSP oligomeric state. These measurements showed that purified MSP in solution is a mixture of monomers and dimers, while solubilized MSP crystals contained only dimers. Size-exclusion chromatography and dynamic light scattering were used to probe the effect of the crystallization conditions on the MSP. Size-exclusion chromatography of concentrated MSP showed the presence of aggregates and monomers, while dilute MSP contained monomers. Dynamic light scattering experiments in the absence, or in the presence of 10–50 mM or 100 mM calcium, yielded calculated molecular mass values of 34 kDa, 48 kDa and 68 kDa, respectively. These changes in the observed molecular mass of the MSP could have been caused by the formation of dimers and higher oligomers and/or significant conformational changes. Based on the results reported in this study, a model is presented which details the effect of oligomeric heterogeneity on the inhibition of MSP crystal growth.

The phycobilisome photosynthetic antenna complex, found in cyanobacteria and red-algae, interacts with proteins expressed specifically to deal with different forms of physiological stress. Under conditions of nutrient starvation, the NblA protein is required for the process that leads to phycobilisome degradation and bleaching of the cells. HspA, a 16.5 kDa heat shock protein expressed in cyanobacterial cells, has been shown to provide functional stability to the phycobilisome during heat stress. We have cloned the genes encoding for these proteins into bacterial expression vectors in order to determine their three-dimensional structures. The resulting recombinant proteins were found to be sparingly soluble, limiting their usefulness in the performance of crystallization experiments. We have developed a novel protocol that utilizes relatively high concentrations of urea to afford sufficient solubility to the protein. This has lead to the successful growth of diffraction quality crystals of these proteins. Complete data sets collected to 2–2.5 Å from crystals of both proteins shows that the crystals are stable, and useful for structure determination. A preliminary structure of the NblA shows that denaturation has not occurred and specific protein-protein interactions have been preserved. We believe that this protocol may be a generally advantageous method to obtain well diffracting crystals of sparingly soluble proteins.

Oxygen-evolving photosystem II reaction centres (RCII) isolated from both spinach and pea have been crystallized. A single crystal form grew from RCII monomers in the presence of nine different three-component mixtures of non-ionic detergents and heptane-1,2,3-triol. The crystals grew as hexagonal rods with dimensions of up to 1 × 0.3 × 0.3 mm. The crystals diffracted to a maximum resolution of 6.5 Å and belong to a hexagonal space group with unit-cell parameters a = 495, b = 495, c = 115 Å,  =  = 90,  = 120°. The growth of a single crystal form in the presence of such a large variety of detergents suggests a very limited range of crystal lattice formation sites in the RCII complex.

Tyrosinase is responsible for the two initial enzymatic steps in the conversion of tyrosine to melanin. Many tyrosinase mutations are the leading cause of albinism in humans, and it is a prominent biotechnology and pharmaceutical industry target. Here we present crystal structures that show that both monophenol hydroxylation and diphenol oxidation occur at the same site. It is suggested that concurrent presence of a phenylalanine above the active site and a restricting thioether bond on the histidine coordinating CuA prevent hydroxylation of monophenols by catechol oxidases. Furthermore, a conserved water molecule activated by E195 and N205 is proposed to mediate deprotonation of the monophenol at the active site. Overall, the structures reveal precise steps in the enzymatic catalytic cycle as well as differences between tyrosinases and other type-3 copper enzymes.

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an α-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.

In recent years, finding alternatives for fossil fuels has become a major concern. One promising solution is microorganism-based bio-photo electrochemical cells (BPECs) that utilize photosynthetic solar energy conversion as an energy source while absorbing CO2 from the atmosphere. It was previously reported that in cyanobacterial-based BPECs, the major endogenous electron mediator that can transfer electrons from the thylakoid membrane photosynthetic complexes and external anodes is NADPH. However, the question of whether the same electron transfer mechanism is also valid for live eukaryotic microalgae, in which NADPH must cross both the chloroplast outer membrane and the cell wall to be secreted from the cell has remained elusive. In this work, we show that NADPH is also the major endogenous electron mediator in the microalgae Dunalliela salina (Ds). We show that the ability of Ds to tolerate high salinity enables the production of a photocurrent that is 5–6 times greater than previously reported for freshwater cyanobacterial-based BPECs in the presence or absence of exogenous electron mediators. Additionally, we show that the electron mediator Vitamin B1 can also function as an electron mediator enhancing photocurrent production. Finally, we show that the addition of both FeCN and NADP+ to Ds has a synergistic effect enhancing the photocurrent beyond the effect of adding each mediator separately.

The molecular architectures of photosynthetic complexes are rapidly becoming available through the power of X-ray crystallography. These complexes are comprised of antenna complexes, which absorb and transfer energy into photochemical reaction centers. Most reaction centers, found in both oxygenic and non-oxygenic species, are connected to transmembrane chlorophyll containing antennas, and the crystal structures of these antennas contain information on the structure of the entire complex as well as clear indications on their modes of functional association. In cyanobacteria and red alga, most of the Photosystem II associated light harvesting is performed by an enormous (3–7 MDa) membrane attached complex called the phycobilisome (PBS). While the crystal structures of many isolated components of different PBSs have been determined, the structure of the entire complex as well as its manner of association with Photosystem II can only be suggested. In this review, the structural information obtained on the isolated components will be described. The structural information obtained from the components provides the basis for the modeled reconstruction of this giant complex.

Clues to designing highly efficient organic solar cells may lie in understanding the architecture of light-harvesting systems and exciton energy transfer (EET) processes in very efficient photosynthetic organisms. Here, we compare the kinetics of excitation energy tunnelling from the intact phycobilisome (PBS) light-harvesting antenna system to the reaction center in photosystem II in intact cells of the cyanobacterium Acaryochloris marina with the charge transfer after conversion of photons into photocurrent in vertically aligned carbon nanotube (va-CNT) organic solar cells with poly(3-hexyl)thiophene (P3HT) as the pigment. We find that the kinetics in electron hole creation following excitation at 600 nm in both PBS and va-CNT solar cells to be 450 and 500 fs, respectively. The EET process has a 3 and 14 ps pathway in the PBS, while in va-CNT solar cell devices, the charge trapping in the CNT takes 11 and 258 ps. We show that the main hindrance to efficiency of va-CNT organic solar cells is the slow migration of the charges after exciton formation.

The initial steps of oxygenic photosynthetic electron transfer occur within photosystem II, an intricate pigment/protein transmembrane complex. Light-driven electron transfer occurs within a multistep pathway that is efficiently insulated from competing electron transfer pathways. The heart of the electron transfer system, composed of six linearly coupled redox active cofactors that enable electron transfer from water to the secondary quinone acceptor QB, is mainly embedded within two proteins called D1 and D2. We have identified a site in silico, poised in the vicinity of the QA intermediate quinone acceptor, which could serve as a potential binding site for redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu) in the cyanobacterium Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically. The Glu thylakoid membranes are able to perform light-dependent reduction of exogenous cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant was also shown to significantly protect the D1 protein from photodamage when isolated thylakoid membranes were illuminated. We have therefore engineered a novel electron transfer pathway from water to a soluble protein electron carrier without harming the normal function of photosystem II.

A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. 1H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc’s distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.

To improve the energy conversion efficiency of solar organic cells, the clue may lie in the development of devices inspired by an efficient light harvesting mechanism of some aquatic photosynthetic microorganisms that are adapted to low light intensity. Consequently, we investigated the pathways of excitation energy transfer (EET) from successive light harvesting pigments to the low energy level inside the phycobiliprotein antenna system of Acaryochloris marina, a cyanobacterium, using a time resolved absorption difference spectroscopy with a resolution time of 200 fs. The objective was to understand the actual biochemical process and pathways that determine the EET mechanism. Anisotropy of the EET pathway was calculated from the absorption change trace in order to determine the contribution of excitonic coupling. The results reveal a new electron energy relaxation pathway of 14 ps inside the phycocyanin component, which runs from phycocyanin to the terminal emitter. The bleaching of the 660 nm band suggests a broader absorption of the terminal emitter between 660 nm and 675 nm. Further, there are trimer depolarization kinetics of 450 fs and 500 fs in high and low ionic strength, respectively, which arise from the relaxation of the β84 and α84 in adjacent monomers of phycocyanin. Under conditions of low ionic strength buffer solution, the evolution of the kinetic amplitude during the depolarization of the trimer is suggestive of trimer conservation within the phycocyanin hexamer. The anisotropy values were 0.38 and 0.40 in high and in low ionic strength, respectively, indicating that there is no excitonic delocalization in the high energy level of phycocyanin hexamers.

The autosomal dominant cerebellar ataxias, referred to as spinocerebellar ataxias in genetic nomenclature, are a rare group of progressive neurodegenerative disorders characterized by loss of balance and coordination. Despite the identification of numerous disease genes, a substantial number of cases still remain without a genetic diagnosis. Here, we report five novel spinocerebellar ataxia genes, FAT2, PLD3, KIF26B, EP300, and FAT1, identified through a combination of exome sequencing in genetically undiagnosed families and targeted resequencing of exome candidates in a cohort of singletons. We validated almost all genes genetically, assessed damaging effects of the gene variants in cell models and further consolidated a role for several of these genes in the aetiology of spinocerebellar ataxia through network analysis. Our work links spinocerebellar ataxia to alterations in synaptic transmission and transcription regulation, and identifies these as the main shared mechanisms underlying the genetically diverse spinocerebellar ataxia types.

Erythrokeratolysis hiemalis (EH) also known as keratolytic winter erythema is a rare autosomal-dominantly inherited disorder of cornification. It manifests with recurrent episodes of skin peeling and palmoplantar erythema, often more evident during the winter season. EH was recently found to be caused by genomic duplications in a non-coding region which regulates CTSB, a gene encoding the cysteine protease Cathepsin B. As a consequence, Cathepsin B expression is increased which in turn causes EH. We aimed at identifying the genetic defect underlying an atypical disorder of cornification in a family featuring severe diffuse transgradient hyperkeratosis involving the palms and soles accompanied by mild diffuse erythema of the palms. Pedigree analysis suggested autosomal dominant inheritance. Whole-exome sequencing revealed a heterozygous missense variant in CTSB. The variant affects a highly conserved residue and is predicted to be pathogenic. Protein modelling indicated that the variant is likely to lead to increased endopeptidase Cathepsin B activity. Accordingly, using a reporter assay expressing the variant, we found that it indeed results in increased Cathepsin B activity. Moreover, Cathepsin B expression was increased in a biopsy sample obtained from the patient. In summary, we report the identification of the first gain-of-function missense variant in CTSB which was found to be associated with an atypical EH-like phenotype.

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0–2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.

The use of photosystem II (PSII) in hybrid bio-photoelectrochemical cells for conversion of solar energy to electrical current is hampered by PSII’s narrow absorption cross-section and the generally poor electrical connection between isolated complex and different anode materials. Here, we merge isolated market-grade spinach PSII with 25 nm cystamine-2,6-dichlorobenzoquinone (cys-DCBQ) modified Au-nanoparticles (PSII–AuNPCys-DCBQ) to obtain one of the highest reported photocurrent values to date (35 mA cm−2 mg−1 chlorophyll), retaining the native oxygen evolution properties of PSII. More than 80% of the PSII in solution assembles stably onto the AuNPCys-DCBQ. Spectroscopic studies show strong functional association in these hybrid particles. Mechanistic investigations reveal a dual role of the quinone-modified AuNPs, harvesting and transference of the excitation energy to PSII (resulting in ∼10-fold enhancement of anodic photocurrent in the 500–550 nm irradiation range) and simultaneously improving the electron transfer from PSII into the graphite anode (by almost 6-fold over the entire visible range of light).

The conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Life on earth depends on photosynthesis, the major mechanism for biological conversion of light energy into chemical energy. Indeed, billions of years of evolution and adaptation to extreme environmental habitats have resulted in highly efficient light-harvesting and photochemical systems in the photosynthetic organisms that can be found in almost every ecological habitat of our world. In harnessing photosynthesis to produce green energy, the native photosynthetic system is interfaced with electrodes and electron mediators to yield bio-photoelectrochemical cells (BPECs) that transform light energy into electrical power. BPECs utilizing plants, seaweeds, unicellular photosynthetic microorganisms, thylakoid membranes or purified complexes, have been studied in attempts to construct efficient and non-polluting BPECs to produce electricity or hydrogen for use as green energy. The high efficiency of photosynthetic light-harvesting and energy production in the mostly unpolluting processes that make use of water and CO2 and produce oxygen beckons us to develop this approach. On the other hand, the need to use physiological conditions, the sensitivity to photoinhibition as well as other abiotic stresses, and the requirement to extract electrons from the system are challenging. In this review, we describe the principles and methods of the different kinds of BPECs that use natural photosynthesis, with an emphasis on BPECs containing living oxygenic photosynthetic organisms. We start with a brief summary of BPECs that use purified photosynthetic complexes. This strategy has produced high-efficiency BPECs. However, the lifetimes of operation of these BPECs are limited, and the preparation is laborious and expensive. We then describe the use of thylakoid membranes in BPECs which requires less effort and usually produces high currents but still suffers from the lack of ability to self-repair damage caused by photoinhibition. This obstacle of the utilization of photosynthetic systems can be significantly reduced by using intact living organisms in the BPEC. We thus describe here progress in developing BPECs that make use of cyanobacteria, green algae, seaweeds and higher plants. Finally, we discuss the future challenges of producing high and longtime operating BPECs for practical use.

Epidermolysis bullosa (EB) refers to a heterogeneous group of rare inherited disorders characterized by skin and mucosal fragility.1 Junctional EB (JEB) is an autosomal recessive (AR) subtype of EB manifesting with widespread skin blistering, mucosal sloughing and, in some cases, nail dystrophy, alopecia and visceral involvement. Pathogenic variants in seven genes have been found to cause JEB, including LAMA3, LAMB3 and LAMC2, encoding laminin 332‐polypeptides; COL17A1, encoding type XVII collagen; ITGA3, encoding integrin α3; and ITGA6 and ITGB4, encoding integrin α6β4 subunits, components of the hemisdesmosome complex. The integrin β4 intracytoplasmic domain interacts with keratin filaments through plectin and type XVII collagen. Biallelic ITGB4 mutations result in recessive JEB with (JEB‐PA, MIM 226730) or without (JEB, MIM 619816) pyloric atresia. In addition, a single heterozygous missense mutation in ITGB4 (c.433G>T, p.Asp145Tyr) has been reported to cause in one family nail dystrophy and late‐onset mild skin fragility with hypergranulation tissue, lacrimal duct obstruction and urethral strictures in some affected individuals.

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (LR8.7), the rod linker (LR), and only one of three rod–core linkers (LRC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the LR protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.

Photoelectrochemical water splitting uses solar power to decompose water to hydrogen and oxygen. Here we show how the photocatalytic activity of thylakoid membranes leads to overall water splitting in a bio-photo-electro-chemical (BPEC) cell via a simple process. Thylakoids extracted from spinach are introduced into a BPEC cell containing buffer solution with ferricyanide. Upon solar-simulated illumination, water oxidation takes place and electrons are shuttled by the ferri/ferrocyanide redox couple from the thylakoids to a transparent electrode serving as the anode, yielding a photocurrent density of 0.5 mA cm−2. Hydrogen evolution occurs at the cathode at a bias as low as 0.8 V. A tandem cell comprising the BPEC cell and a Si photovoltaic module achieves overall water splitting with solar to hydrogen efficiency of 0.3%. These results demonstrate the promise of combining natural photosynthetic membranes and man-made photovoltaic cells in order to convert solar power into hydrogen fuel.

Pachyonychia congenita (PC) refers to a group of autosomal dominant disorders caused by mutations in five keratin genes (KRT16,KRT6A,KRT17,KRT6B or KRT6C). Current disease classification is based on the gene harbouring disease‐causing variants. We harnessed the International Pachyonychia Congenita Research Registry (IPCRR) containing both clinical and molecular data on patients with PC worldwide, to identify genetic variants predicting disease severity. We ascertained 815 individuals harbouring keratin mutations registered in the IPCRR. We looked for statistically significant associations between genetic variants and clinical manifestations in a subgroup of patients carrying mutations found in at least 10% of the cohort. Data were analysed using χ2 and Kruskal–Wallis tests. We identified five mutations occurring in at least 10% of the patients registered in the IPCRR. The KRT16 p.L132P mutation was significantly associated with younger age of onset, presence of palmar keratoderma oral leucokeratosis and a higher number of involved nails. By contrast, the KRT16 p.N125S and p.R127C mutations resulted in a milder phenotype featuring a decreased number of involved nails and older age of onset. Patients carrying the p.N125S mutation were less likely to develop palmar keratoderma while p.R127C was associated with an older age of palmoplantar keratoderma onset. Moreover, the KRT17 p.L99P mutation resulted in an increased number of involved fingernails and patients demonstrating 20‐nail dystrophy, while the opposite findings were observed with KRT17 p.N92S mutation. We have identified novel and clinically useful genetic predictive variants in the largest cohort of patients with PC described to date.

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the β4-β5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

Photosystem II (PSII) is the only enzyme that catalyzes light-induced water oxidation, the basis for its application as a biophotoanode in various bio-photovoltaics and photo-bioelectrochemical cells. However, the absorption spectrum of PSII limits the quantum efficiency in the range of visible light, due to a gap in the green absorption region of chlorophylls (500–600 nm). To overcome this limitation, we have stabilized the interaction between PSII and Phycobilisomes (PBSs) – the cyanobacterial light harvesting complex, in vitro. The PBS of three different cyanobacteria (Acaryochloris marina, Am, Mastigocladus laminosus, ML, and Synechocystis sp. PCC 6803, Syn) are analyzed for their ability to transfer energy to Thermosynechococcus elongatus (Te) PSII by fluorescence spill-over and photo-current action spectra. Integration of the PBS–PSII super-complexes within an Os-complex-modified hydrogel on macro-porous indium tin oxide electrodes (MP-ITO) resulted in notably improved, wavelength dependent, incident photon-to-electron conversion efficiencies (IPCE). IPCE values in the green gap were doubled from 3% to 6% compared to PSII electrodes without PBS and a maximum IPCE up to 10.9% at 670 nm was achieved.

Tyrosinase is a type 3 copper enzyme with great potential for production of commercially valuable diphenols from monophenols. However, the use of tyrosinase is limited by its further oxidation of diphenols to quinones. We recently determined the structure of the Bacillus megaterium tyrosinase revealing a residue, V218, which we proposed to take part in positioning of substrates within the active site. In the structure of catechol oxidase from Ipomoea batatas, the lack of monophenolase activity was attributed to the presence of F261 near CuA. Consequently, we engineered two variants, V218F and V218G. V218F was expected to have a decreased monophenolase activity, due to the bulky residue extending into the active site. Surprisingly, both V218F and V218G exhibited a 9- and 4.4-fold higher monophenolase/diphenolase activity ratio, respectively. X-ray structures of variant V218F display a flexibility of the phenylalanine residue along with an adjacent histidine, which we propose to be the source of the change in activity ratio.

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.

Cpn60-2 is a member of a unique family of putative molecular chaperones homologous to GroEL (Cpn60) but of unknown function and found only in Mycobacterium tuberculosis and closely related species. Cpn60-2 has mainly been studied for its strong immunogenity. Here, the purification, crystallization and preliminary crystallographic analysis of M. tuberculosis Cpn60-2 are reported. The crystals belong to space group P2, with unit-cell parameters a = 57, b = 115.5, c = 81.5 Å, [beta] = 95.5°, and contain a dimer in the asymmetric unit. The crystals diffract to 4.0 Å using a Cu rotating-anode X-ray generator.

Light induces an irreversible modification of the photosystem II reaction center (RCII) affecting specifically one of its major components, the D1 protein (Ohad, I., Adir, N., Koike, H., Kyle, D. J., and Inoue, Y. I. (1990) J. Biol. Chem. 265, 1972-1979) which is degraded and replaced continuously (turnover). The turnover rate of D1 is related to light intensity. Evidence is presented showing that RCII translocates from the site of damage in the grana (appressed) domain of the chloroplast membranes to unappressed membrane domains where the D1 precursor protein (pD1) is translated and becomes integrated into RCII. Several forms of RCII (a, a*, and b) were identified on the basis of their electrophoretic mobility. pD1 was found only in the a and b forms in the unappressed membranes. Processing of pD1 occurs after its integration into RCII. Mature D1 appeared mostly in the a form of RCII and following its translocation to the appressed membrane domains also in the a* form. Thus the light intensity-dependent synthesis of D1 protein is related to the availability of modified RCII which serves as an acceptor for pD1. The shuttling of RCII between the two membrane domains may represent a control mechanism of thylakoid membrane protein synthesis.

We applied a femtosecond flash method, using induced transient absorption changes, to obtain a time-resolved view of excitation energy transfer in intact phycobilisomes of Thermosynechococcus vulcanus at room temperature. Our measurement of an excitation energy transfer rate of 888 fs in phycobilisomes shows the existence of ultrafast kinetics along the phycocyanin rod subcomplex to the allophycocyanin core that is faster than expected for previous excitation energy transfer based on Förster theory in phycobilisomes. Allophycocyanin in the core further transfers energy to the terminal emitter(s) in 17 ps. In the phycobilisome, rod doublets composed of hexameric phycocyanin discs and internal linker proteins are arranged in a parallel fashion, facilitating direct rod–rod interactions. Excitonic splitting likely drives rod absorption at 635 nm as a result of strong coupling between β84 chromophores (20 ± 1 Å) in adjacent hexamers. In comparison to the absorbance of the phycobilisome antenna system of the cyanobacterium Acaryochloris marina, which possesses a single rod structure, the linkers in T. vulcanus rods induce a 17 nm red shift in the absorbance spectrum. Furthermore, the kinetics of 888 fs indicates that the presence of the linker protein induces ultrafast excitation energy transfer between phycocyanin and allophycocyanin inside the phycobilisome, which is faster than all previous excitation energy transfer in phycobilisome subunits or sub-complexes reported to date.

Oxygenic photosynthetic organisms perform solar energy conversion of water and CO2 to O2 and sugar at a broad range of wavelengths and light intensities. These cells also metabolize sugars using a respiratory system that functionally overlaps the photosynthetic apparatus. In this study, we describe the harvesting of photocurrent used for hydrogen production from live cyanobacteria. A non-harmful gentle physical treatment of the cyanobacterial cells enables light-driven electron transfer by an endogenous mediator to a graphite electrode in a bio-photoelectrochemical cell, without the addition of sacrificial electron donors or acceptors. We show that the photocurrent is derived from photosystem I and that the electrons originate from carbohydrates digested by the respiratory system. Finally, the current is utilized for hydrogen evolution on the cathode at a bias of 0.65 V. Taken together, we present a bio-photoelectrochemical system where live cyanobacteria produce stable photocurrent that can generate hydrogen.

Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis. The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (ΔHa) and allows the determination of the binding constant (free energy), entropy, and stoichiometry. In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6. The change in the heat capacity of binding (ΔCp = ΔH/ΔT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures. Because hydrophobic stacking interactions are associated with negative ΔCp, the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme. The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis.

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77 K. The study demonstrates that if complexes are stabilized by high phosphate (900 mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50 mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.

Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77 K revealed two emission bands at ∼690 nm and 725 nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725 nm emission band is associated with low energy Chls a with absorption band clearly resolved at ∼710 nm at 77 K. In summary, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.

The light-induced inactivation of the photochemical reaction center II (RCII) of oxygenic chloroplasts (photoinhibition) was investigated in cells and isolated thylakoids of the green alga Chlamydomonas reinhardtii. The process is resolved into a reversible conformational change followed by an irreversible modification of RCII D1 protein. The light-induced changes in vivo persisted in isolated thylakoids. The first step is characterized by (i) destabilization of the secondary acceptor semiquinone anion, Q-B, bound to the D1 protein. This is demonstrated by a reduction in the activation energy of S2,3Q-B charge recombination as measured by the thermoluminescence technique; and (ii) a rise in the intrinsic fluorescence and a decrease of the maximal fluorescence. Unoccupancy of the QB site by plastoquinone partially protected RCII against the light-induced destabilization of Q-B. The extent of charge separation (P+680Q-A) was not affected. However, the slow phase (microsecond) of P+680 dark reduction increased, and the amplitude of signal II was reduced by 20-30%, indicating that in a fraction of RCII, electron donation from Z to P+680 was impaired without losing primary photochemistry. This modification correlates with the irreversible change in D1 protein resulting in the formation of a trypsin-resistant fragment of 16 kDa detected in D1 isolated from light-exposed cells. The change in the Q-B stability could allow charge equilibration with QA and thus explain the rise in the intrinsic fluorescence level and reduction of electron flow to plastoquinone. The change in the lifetime of P+680 can account for further reduction in electron flow (photo-inhibition). The irreversible light-dependent modification of D1 may serve as the signal for its degradation and replacement by a newly synthesized molecule (turnover).

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in α helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the Km rather than changes in the kcat values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA–DNA substrate.

Despite the high homology between human immunodeficiency virus type-1 (HIV-1) and human immunodeficiency virus type-2 (HIV-2) reverse transcriptases (RTs), the ribonuclease H (RNase H) level of HIV-2 RT is lower than that of HIV-1 RT, while the DNA polymerase of both RTs is similar. We conducted mutagenesis of HIV-2 RT Gln294 (shown to control the RNase H activity level when modified to a Pro in the smaller p54 subunit and not in the larger p68 subunit) to various residues, and assayed the activities of all mutants. All exhibited an RNase H that is higher than the wild-type (WT) HIV-2 RT level, although the DNA polymerase of all mutants equals WT HIV-2 RT level. These results represent a unique case, where every mutation induces an increase rather than a decrease in an enzyme’s activity.

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway.

The fibroblast growth factor (FGF) family plays a key role in a multitude of physiological and pathological processes. The activities of FGFs are mediated by a family of tyrosine kinase receptors, designated FGFRs. The mechanism by which FGFs induce receptor activation is controversial. Despite their structural similarity, FGFs display distinct receptor binding characteristics and cell type specificity. Previous studies with FGF-2 identified a low affinity receptor binding site that is located within a loop connecting its 9th and 10th β-strands. The corresponding residues in the other family members are highly variable, and it was proposed that the variability might confer on FGFs unique receptor binding characteristics. We studied the role of this loop in FGF-7 by both site-directed mutagenesis and loop replacement. Unlike the other members of the FGF family, FGF-7 recognizes only one FGFR isoform and is, therefore, ideal for studies of how the specificity in the FGF-FGFR interaction is conferred at the structural level. Point mutations in the loop of FGF-7 did not change receptor binding affinity but resulted in reduced mitogenic potency and reduced ability to induce receptor-mediated phosphorylation events. These results suggest that the loop of FGF-7 fulfills the role of low affinity binding site required for receptor activation. The observation that it is possible to uncouple FGF-7 receptor binding and biological activity favors a bivalent model for FGFR dimerization, and it may be clinically relevant to the design of FGF-7 antagonists. Reciprocal loop replacement between FGF-7 and FGF-2 had no effect on their known receptor binding affinities nor did it alter their known specificity in eliciting a mitogenic response. In conclusion, these results suggest that, despite the diversity in the loop structure of FGF-2 and FGF-7, the loop has a similar function in both growth factors.

The aims of this study were to (1) characterize the clinical phenotype, (2) define the causative mutation, and (3) correlate the clinical phenotype with genotype in a large consanguineous Arab family with myotonia congenita. Twenty-four family members from three generations were interviewed and examined. Genomic DNA was extracted from peripheral blood samples for sequencing the exons of the CLCN1 gene. Twelve individuals with myotonia congenita transmitted the condition in an autosomal dominant manner with incomplete penetrance. A novel missense mutation [568GG>TC (G190S)] was found in a dose-dependent clinical phenotype. Although heterozygous individuals were asymptomatic or mildly affected, the homozygous individuals were severely affected. The mutation is a glycine-to-serine residue substitution in a well-conserved motif in helix D of the CLC-1 chloride channel in the skeletal muscle plasmalemma. A novel mutation, 568GG>TC (G190S) in the CLCN1 gene, is responsible for autosomal dominant myotonia congenita with a variable phenotypic spectrum. Muscle Nerve, 2009

Previous studies have shown that live cyanobacteria can produce photocurrent in bio-photoelectrochemical cells (BPECs) that can be exploited for clean renewable energy production. Electron transfer from cyanobacteria to the electrochemical cell was proposed to be facilitated by small molecule(s) mediator(s) whose identity (or identities) remain unknown. Here, we elucidate the mechanism of electron transfer in the BPEC by identifying the major electron mediator as NADPH in three cyanobacterial species. We show that an increase in the concentration of NADPH secreted into the external cell medium (ECM) is obtained by both illumination and activation of the BPEC. Elimination of NADPH in the ECM abrogates the photocurrent while addition of exogenous NADP+ significantly increases and prolongs the photocurrent production. NADP+ is thus the first non-toxic, water soluble electron mediator that can functionally link photosynthetic cells to an energy conversion system and may serve to improve the performance of future BPECs.

Normophosphatemic familial tumoral calcinosis (NFTC) is an autosomal recessive disorder characterized by calcium deposition in skin and mucosae and associated with unremitting pain and life-threatening skin infections. A homozygous missense mutation (p.K1495E), resulting in SAMD9 protein degradation, was recently shown to cause NFTC in five families of Jewish-Yemenite origin. In this study, we evaluated another Jewish-Yemenite NFTC kindred. All patients were compound heterozygous for two mutations in SAMD9: K1495E and a previously unreported nonsense mutation, R344X, predicted to result in a markedly truncated molecule. Screening of unaffected population-matched controls revealed heterozygosity for K1495E and R344X only in individuals of Jewish-Yemenite ancestry, but not in more than 700 control samples of other origins, including 93 non-Jewish Yemenite. These data may be suggestive of positive selection, considering the rarity of NFTC and the small size of the Jewish-Yemenite population; alternatively, they may reflect genetic drift or the effect of a population-specific modifier trait. Calcifications in NFTC generally develop over areas subjected to repeated trauma and are associated with marked inflammatory manifestations, indicating that SAMD9 may play a role in the inflammatory response to tissue injury. We therefore assessed the effect of cellular stress and tumor necrosis factor-α (TNF-α), a potent pro-inflammatory cytokine, on SAMD9 gene expression. Whereas exogenous hydrogen peroxide and heat shock did not affect SAMD9 transcription, osmotic shock was found to markedly upregulate SAMD9 expression. In addition, incubation of endothelial cells with TNF-α caused a dose-related, p38-dependant increase in SAMD9 expression. These data link NFTC and SAMD9 to the TNF-α signaling pathway, suggesting a role for this system in the regulation of extra-osseous calcification.

Olmsted syndrome (OS; MIM 614594) is a rare genodermatosis featuring symmetric and mutilating palmoplantar keratoderma (PPK) and periorificial keratotic plaques (Olmsted, 1927). Diffuse alopecia, onychodystrophy, oral leucokeratosis, corneal lesions, and pseudoainhum may be associated as well (Mevorah et al., 2005; Lai-Cheong et al., 2012). Extracutaneous manifestations are uncommon and include deafness, mental retardation, joint laxity, osteopenia, and osteolysis, secondary infections, and squamous cell carcinoma developing in areas of PPK (Mevorah et al., 2005).

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5–10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.

Erythrokeratolysis hiemalis (EH; MIM148370), also known as keratolytic winter erythema, is a rare autosomal dominant disorder of cornification featuring recurrent episodes of erythema, hyperkeratosis, and peeling of palms and soles, with typical worsening during the winter months.1 Although EH is common in the South African population, with a reported prevalence of 1 in 7000 as a result of a founder mutation,1 it has also been reported in individuals of German,1 Danish2 and Norwegian3 origin. Linkage analysis initially mapped the EH trait to chromosome 8p23.1−p22.1 Several studies then searched for relevant candidate genes in this specific region, but failed to identify causative mutations in the coding region of FDFT1 and CSTB.4, 5 More recently, EH was found to result from genomic duplications involving a noncoding region that regulates CTSB expression.3 CTSB encodes the cysteine protease cathepsin B. EH‐causing genomic duplications were found to result in increased expression of cathepsin B in patient skin.3

In the current study, we aimed to delineate the genetic basis of an EH‐like phenotype in a patient with diffuse and erythematous palmoplantar keratoderma transgrediens.

Light is a common source of energy in sustainable technologies for photocurrent generation. To date, in such light-harvesting applications, the excited electrons generate the photocurrent. Here, we introduce a new mechanism for photocurrent generation that is based on excited state proton transfer (ESPT) of photoacids and photobases that can donate or accept a proton, respectively, but only after excitation. We show that the formed ions following ESPT can either serve as electron donors or acceptors with the electrodes, or modify the kinetics of mass transport across the diffuse layer, both resulting in photocurrent generation. We further show that control of the current polarity is obtained by switching the irradiation between the photoacid and the photobase. Our study represents a new approach in photoelectrochemistry by introducing ESPT processes, which can be further utilized in light-responsive energy production or energy storage.

The effect of unoccupancy of the QB site by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QB site are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QB charge recombination, rise in in­ trinsic fluorescence and loss of variable fluorescence. These parameters relate to functions in­ volving the reaction center II D1 protein. The light-induced degradation of D1 in the mutant cells was also considerably reduced, with a t 1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D1 protein are related and require occupancy of the QB site by PQ and its light-driven reduction.

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II ‘life cycle.’

The increasing concern about future environmental disasters as a result of global climate change is driving the efforts to develop novel clean energy technologies that will replace combustible fuels. A promising approach is the utilization of native photosynthesis as an electron source in bio-electrochemical cells. Over the past few years, there have been numerous descriptions of technologies for electrical current production using isolated photosynthetic complexes or membranes, intact microorganisms, or microorganism tissues in different electrochemical setups. In this review, we summarize these methods, and address the key factors that influence photocurrent generation and how can they be improved. Finally, we discuss the potential of photosynthetic-based bioelectricity production to become a real applicative solution that will be able to compete with existing technologies such as solar cells.

Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.

Photosynthesis is driven by the absorption of light by arrays of pigments bound within protein complexes called antennas, followed by the efficient transfer of energy to the photochemical reaction centers. Cyanobacteria, red algae and cyanelles contain phycobilisomes (PBS) as their major antenna complex. The PBS is an extremely large (3–7 MDa), multi-layered complex bound to the stromal side of the photosynthetic membrane. In this review, we will describe the important structural and functional characteristics of the phycobilisome complex experimentally obtained over the past 40 years, especially in relation to the phycobilisomes unique absorption characteristics and its ability to self-assemble and disassemble.

Manganese is recruited in microorganisms by way of ABC-type transporter systems. Here, the expression, purification and preliminary crystallographic analysis of a soluble form of the MntC solute-binding protein component of the MntABC manganese-import system from the cyanobacterium Synechococystis sp. PCC 6803 is reported. The protein (321 amino-acid residues) was expressed exclusively in inclusion bodies, which required unfolding and refolding in the presence of manganese prior to purification. The purified protein was crystallized in the presence of PEG and zinc. The crystals belong to space group P6222, with unit-cell parameters a = b = 128.1, c = 90.0 Å and a single molecule in the asymmetric unit. The crystals diffract to 2.6 Å under cryoconditions using synchrotron radiation.

Here, we show that it is possible to harvest photocurrent directly from unprocessed plant tissues from terrestrial or aquatic environments in bio-photoelectrochemical cells (BPECs) and use the current to produce molecular H2. The source of electrons is shown to originate from the Photosystem II water-oxidation reaction and utilizes exported mediating molecules, especially NADPH. The photocurrent production is dependent on the concentration of the photosynthetic complexes, as an increase in total chlorophyll and oxygen evolution rates in the leaves lead to increased photocurrent rates. The permeability of the outer leaf surface is another important factor in photocurrent harvesting. Different tissues produce photocurrent densities in the range of ∼1–10 mA/cm2 which is significantly higher than microorganism-based BPECs. The relatively high photocurrent and the simplicity of the plants BPEC may pave the way toward the development of future applicative photosynthetic based energy technologies.

Using single-crystal x-ray diffraction, we found a formerly unknown twin form in calcite crystals grown from solution to which a mollusc shell-derived 17-kDa protein, Caspartin, was added. This intracrystalline protein was extracted from the calcitic prisms of the Pinna nobilis shells. The observed twin form is characterized by the twinning plane of the (108)-type, which is in addition to the known four twin laws of calcite identified during 150 years of investigations. The established twin forms in calcite have twinning planes of the (001)-, (012)-, (104)-, and (018)-types. Our discovery provides additional evidence on the crucial role of biological macromolecules in biomineralization.

A 40 kD protein has been extracted from the biomineral matrix of the calcium carbonate gastropod shell of Strombus decorus persicus. The protein was isolated by decalcification and ion exchange HPLC. We have named this protein ACLS40, i.e., aragonite crossed-lamellar structure protein. A partial sequence of the isolated ACLS40 and amino acid analysis both indicate that it does not belong to the family of very acidic proteins, i.e., rich in aspartic and glutamic residues. The shell-extracted protein shows the ability to stabilize calcium carbonate in vitro, in the form of thermodynamically unstable vaterite polymorph, and to inhibit the growth of calcite.

Writer’s cramp (WC) is a task-specific focal dystonia that occurs selectively in the hand and arm during writing. Previous studies have shown a role for genetics in the pathology of task-specific focal dystonia. However, to date, no causal gene has been reported for task-specific focal dystonia, including WC. In this study, we investigated the genetic background of a large Dutch family with autosomal dominant‒inherited WC that was negative for mutations in known dystonia genes. Whole exome sequencing identified 4 rare variants of unknown significance that segregated in the family. One candidate gene was selected for follow-up, Calcium Voltage-Gated Channel Subunit Alpha1 H, CACNA1H, due to its links with the known dystonia gene Potassium Channel Tetramerization Domain Containing 17, KCTD17, and with paroxysmal movement disorders. Targeted resequencing of CACNA1H in 82 WC cases identified another rare, putative damaging variant in a familial WC case that did not segregate. Using structural modelling and functional studies in vitro, we show that both the segregating p.Arg481Cys variant and the non-segregating p.Glu1881Lys variant very likely cause structural changes to the Cav3.2 protein and lead to similar gains of function, as seen in an accelerated recovery from inactivation. Both mutant channels are thus available for re-activation earlier, which may lead to an increase in intracellular calcium and increased neuronal excitability. Overall, we conclude that rare functional variants in CACNA1H need to be interpreted very carefully, and additional studies are needed to prove that the p.Arg481Cys variant is the cause of WC in the large Dutch family.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 Å resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

Photosynthetic organisms harvest light energy, utilizing the absorption and energy-transfer properties of protein-bound chromophores. Controlling the harvesting efficiency is critical for the optimal function of the photosynthetic apparatus. Here, we show that the cyanobacterial light-harvesting antenna complex may be able to regulate the flow of energy to switch reversibly from efficient energy conversion to photoprotective quenching via a structural change. We isolated cyanobacterial light-harvesting proteins, phycocyanin and allophycocyanin, and measured their optical properties in solution and in an aggregated-desiccated state. The results indicate that energy band structures are changed, generating a switch between the two modes of operation, exciton transfer and quenching, achieved without dedicated carotenoid quenchers. This flexibility can contribute greatly to the large dynamic range of cyanobacterial light-harvesting systems.

Quantum nano-structures are likely to become primary elements of future devices. However, there are a number of significant scientific challenges to real world applications of quantum devices. These include de-coherence that erodes operation of a quantum device and control issues. In nature, certain processes have been shown to use quantum mechanical processes for overcoming these barriers. One well-known example is the high energy transmission efficiency of photosynthetic light harvesting complexes. Utilizing such systems for fabricating nano-devices provides a new approach to creating self-assembled nano-energy guides. In this study, we use isolated phycocyanin (PC) proteins that can self-assemble into bundles of nanowires. We show two methods for controlling the organization of the bundles. These nanowires exhibit long range quantum energy transfer through hundreds of proteins. Such results provide new efficient building blocks for coupling to nano-devices, and shed light on distribution and the efficiency of energy transfer mechanisms in biological systems and its quantum nature.

Harvesting an electrical current from biological photosynthetic systems (live cells or isolated complexes) is typically achieved by immersion of the system into an electrolyte solution. In this study, we show that the aqueous solution found in the tissues of succulent plants can be used directly as a natural bio-photo electrochemical cell. Here, the thick water-preserving outer cuticle of the succulent Corpuscularia lehmannii serves as the electrochemical container, the inner water content as the electrolyte into which an iron anode and platinum cathode are introduced. We produce up to 20 μA/cm2 bias-free photocurrent. When 0.5 V bias is added to the iron anode, the current density increases ∼10-fold, and evolved hydrogen gas can be collected with a Faradaic efficiency of 2.1 and 3.5% in dark or light, respectively. The addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits the photocurrent, indicating that water oxidation is the primary source of electrons in the light. Two-dimensional fluorescence measurements show that NADH and NADPH serve as the major mediating electron transfer molecules, functionally connecting photosynthesis to metal electrodes. This work presents a method to simultaneously absorb CO2 while producing an electrical current with minimal engineering requirements.

Striate palmoplantar keratoderma (SPPK) belongs to a group of keratinization disorders characterized by marked thickening of the palms and soles. It is characterized by longitudinal hyperkeratotic lesions extending along each finger to the palm and is most commonly caused by heterozygous nonsense or frameshift mutations in the DSG1 gene, encoding desmoglein 1 (DSG1). In this study, we aimed at identifying the genetic basis underlying SPPK in a patient referred to our clinic. Using direct sequencing we discovered that the patient carries an heterozygous missense mutation in DSG1, c.254A>G; p.Y85C. The mutation was not detected in any public databases, was found to affect a highly conserved residue and was predicted to be highly deleterious. To assess the consequences of the mutation, we modeled the DSG1 mutation at the protein level using three designated software. All software predicted the mutation to compromise DSG1 adhesive function by disrupting DSG1 dimerization, thus resulting in functional haploinsufficiency. We describe here, to our knowledge, the first case of SPPK resulting from an heterozygous missense (as opposed to nonsense or frameshift) mutation in the DSG1 gene. This finding is not only of importance for the diagnostics of SPPK but also emphasizes the importance of DSG1-mediated adhesive function in palmoplantar skin.

The pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3, as a prospective therapeutic candidate for the treatment of VL.

Phycocyanin is one of the two phycobiliproteins always found in the Phycobilisome antenna complex. It is always situated at the ends of the peripheral rods, adjacent to the core cylinders composed of allophycocyanin. The basic phycocyanin monomer is an (αβ) dimer of globin-like subunits with three covalently linked phycocyanobilin cofactors. Monomers assemble further into trimers, hexamers, and rods which include non-pigmented linker proteins. Upon isolation in low ionic strength solution, rods quickly disintegrate into phycocyanin trimers, which lose contacts with other phycobiliproteins and with the linker proteins. The trimers, however, are quite stable and only the presence of high concentrations of chaotropic agents (such as urea), very acidic solutions, or elevated temperatures induces monomerization, followed by separation between the subunits. We have recently determined the crystal structures of phycocyanin from the thremophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 or 4 M urea, and shown that 4 M urea monomerizes the phycocyanin trimers. In this paper, we will describe the phycocyanin structures in 2 and 4 M urea more completely. By mapping out the urea positions, we describe the structural elements within the trimeric interaction interface that may be interrupted by the presence of 4 M urea. In addition, we also identify what are the structural characteristics that prevent 4 M urea from inducing subunit dissociation.

The structural features enabling carotenoid translocation between molecular entities in nature is poorly understood. Here, we present the three-dimensional X-ray structure of an expanded oligomeric state of the C-terminal domain homolog (CTDH) of the orange carotenoid protein, a key water-soluble protein in cyanobacterial photosynthetic photo-protection, at 2.9 Å resolution. This protein binds a canthaxanthin carotenoid ligand and undergoes structural reorganization at the dimeric level, which facilitates cargo uptake and delivery. The structure displays heterogeneity revealing the dynamic nature of its C-terminal tail (CTT). Molecular dynamics (MD) simulations based on the CTDH structures identified specific residues that govern the dimeric transition mechanism. Mutagenesis based on the crystal structure and these MD simulations then confirmed that these specific residues within the CTT are critical for carotenoid uptake, encapsulation and delivery processes. We present a mechanism that can be applied to other systems that require cargo uptake.

The major light harvesting antenna in all cyanobacterial species is the phycobilisome (PBS). The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single four-hexamer rod. We have determined the crystal structure of phycocyanin (AmPC), the major component of the A. marina PBS (AmPBS) to 2.1 Å. The basic unit of the AmPC is a heterodimer of two related subunits (α and β), and we show that the asymmetric unit contains a superposition of two α and two β isoforms, the products of the simultaneous expression of different genes. This is the first time to our knowledge that isolated proteins crystallized with such identifiable heterogeneity. We believe that the presence of the different isoforms allows the AmPBS to have a significant bathochromic shift in its fluorescence emission spectrum, allowing, in the total absence of allophycocyanin, a better overlap with absorption of the chlorophyll d-containing reaction centers. We show that this bathochromic shift exists in intact AmPBS as well as in its disassembled components, thus suggesting that AmPC can efficiently serve as the AmPBS terminal emitter.

A recently reported family of soluble cyanobacterial carotenoproteins, homologs of the C-terminal domain (CTDH) of the photoprotective Orange Carotenoid Protein, is suggested to mediate carotenoid transfer from the thylakoid membrane to the Helical Carotenoid Proteins, which are paralogs of the N-terminal domain of the OCP. Here we present the three-dimensional structure of a carotenoid-free CTDH variant from Anabaena (Nostoc) PCC 7120. This CTDH contains a cysteine residue at position 103. Two dimer-forming interfaces were identified, one stabilized by a disulfide bond between monomers and the second between each monomer’s β-sheets, both compatible with small-angle X-ray scattering data and likely representing intermediates of carotenoid transfer processes. The crystal structure revealed a major positional change of the C-terminal tail. Further mutational analysis revealed the importance of the C-terminal tail in both carotenoid uptake and delivery. These results have allowed us to suggest a detailed model for carotenoid transfer via these soluble proteins.

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2–5 cylinders surrounded by 6–8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4 Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.

Treatment of Chlamydomonas reinhardtii thylakoids with cross-linking reagents including glutaraldehyde causes polymerization of all thylakoid polypeptides, but not of the reaction center II polypeptide D1 unless the thylakoids are presolubilized by octyl beta-D-glucoside (Adir, N., and Ohad, I. (1986) Biochim. Biophys. Acta 850, 264-274). The results presented here show that this is a general property of D1 as it can be demonstrated in thylakoids of cyanophytes, Dasicladaceae, green algae, and C3 and C4 plants. Solubilization of the membranes by ionic detergents, deoxycholate, lauryl sucrose, or dodecyl beta-D-maltoside is not effective in inducing cross-linking of the D1 polypeptides by glutaraldehyde. The most effective alkyl glucosides were those with 7-9 carbon alkyl chains. The same behavior toward glutaraldehyde was exhibited by the unprocessed D1 precursor and by the palmitoylated D1 protein. Based on the refractility of the D1 protein to cross-linking reagents, a procedure was developed for its isolation from cross-linked thylakoids by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Isolated D1 retained its behavior toward cross-linking by glutaraldehyde and generated tryptic fragments similar to those obtained following trypsin treatment of intact thylakoids. Denaturation of isolated D1 protein by acetone facilitates cross-linking by glutaraldehyde and extensive degradation by trypsin. The photosystem II polypeptides are differentially cross-linked with increasing concentrations of glutaraldehyde, the most susceptible being the 28- and 23-kDa components of the light-harvesting chlorophyll a-b protein complex and the core complex 44- and 51-kDa polypeptides, and the least affected being the cytochrome b559, the D2 protein, and a 24-kDa component of the light-harvesting chlorophyll a-b protein complex. These results reflect the relative position and interaction of the photosystem II polypeptides within the complex and suggest that strong and specific hydrophobic interactions may be responsible for the tight and stable conformation of D1. This may be based mostly on the conserved amino acid sequences of D1 and possibly plays a role in the process of D1 integration and removal from the reaction center during its light-dependent turnover.

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (∼6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via “structural mimicry” due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has been determined by molecular replacement to 2.5 Å resolution. The crystal belongs to space group R32 with cell parameters a = b = 188.43 Å, c = 61.28 Å, α = β = 90 °, γ = 120 °, with one (αβ) monomer in the asymmetric unit. The structure has been refined to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 Å. The crystals were grown from phycocyanin (αβ)3 trimers that form (αβ)6 hexamers in the crystals, in a fashion similar to other phycocyanins. Comparison of the primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella diplsiphon and the thermophilic Mastigocladus laminosus were performed. We show that each level of assembly of oligomeric phycocyanin, which leads to the formation of the phycobilisome structure, can be stabilized in thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each association interface. In addition, a significant shift in the position of ring D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin, enables the formation of important polar interactions at both the (αβ) monomer and (αβ)6 hexamer association interfaces.

The heptameric COPI coat (coatomer) plays an essential role in vesicular transport in the early secretory system of eukaryotic cells. While the structures of some of the subunits have been determined, that of the δ-COP subunit has not been reported to date. The δ-COP subunit is part of a subcomplex with structural similarity to tetrameric clathrin adaptors (APs), where δ-COP is the structural homologue of the AP μ subunit. Here, the crystal structure of the μ homology domain (MHD) of δ-COP (δ-MHD) obtained by phasing using a combined SAD–MR method is presented at 2.15 Å resolution. The crystallo­graphic asymmetric unit contains two monomers that exhibit short sections of disorder, which may allude to flexible regions of the protein. The δ-MHD is composed of two subdomains connected by unstructured linkers. Comparison between this structure and those of known MHD domains from the APs shows significant differences in the positions of specific loops and β-sheets, as well as a more general change in the relative positions of the protein subdomains. The identified difference may be the major source of cargo-binding specificity. Finally, the crystal structure is used to analyze the potential effect of the I422T mutation in δ-COP previously reported to cause a neurodegenerative phenotype in mice.

Receptor binding specificity is an essential element in regulating the diverse activities of fibroblast growth factors (FGFs). FGF7 is ideal to study how this specificity is conferred at the structural level, as it interacts exclusively with one isoform of the FGF-receptor (FGFR) family, known as FGFR2IIIb. Previous mutational analysis suggested the importance of the β4/β5 loop of FGF7 in specific receptor recognition. Here a theoretical model of FGFR2IIIb/FGF7 complex showed that this loop interacts with the FGFR2IIIb unique exon. In addition, the model revealed new residues that either directly interact with the FGFR2IIIb unique exon (Asp63, Leu142) or facilitate this interaction (Arg65). Mutations in these residues reduced both receptor binding affinity and biological activity of FGF7. Altogether, these results provide the basis for understanding how receptor-binding specificity of FGF7 is conferred at the structural level.

Cyanobacteria and red-algae share a common light-harvesting complex which is different than all other complexes that serve as photosynthetic antennas – the Phycobilisome (PBS). The PBS is found attached to the stromal side of thylakoid membranes, filling up most of the gap between individual thylakoids. The PBS self assembles from similar homologous protein units that are soluble and contain conserved cysteine residues that covalently bind the light absorbing chromophores, linear tetra-pyrroles. Using similar construction principles, the PBS can be as large as 16.8 MDa (68×45×39nm), as small as 1.2 MDa (24 × 11.5 × 11.5 nm), and in some unique cases smaller still. The PBS can absorb light between 450 nm to 650 nm and in some cases beyond 700 nm, depending on the species, its composition and assembly. In this review, we will present new observations and structures that expand our understanding of the distinctive properties that make the PBS an amazing light harvesting system. At the end we will suggest why the PBS, for all of its excellent properties, was discarded by photosynthetic organisms that arose later in evolution such as green algae and higher plants.

A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized. The crystals belong to the P63 space group with cell dimensions of 153 Å × 153 Å × 59 Å with a single (αβ) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 Å. The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (R free = 27.8%). The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC612 structure shows that the α subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnβ72 residue is not methylated, explaining the blue-shift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core.

Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities found that the alga employs all three known photoinhibition protection mechanisms: (i) massive reduction of the PSII antenna size; (ii) accumulation of protective carotenoids; and (iii) very rapid repair of photodamaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms, and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.

Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNAAsp methylation, the high levels suggested that Ehmeth-mediated tRNAAsp methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.

Tyrosinase belongs to the type 3 copper enzyme family, containing a dinuclear copper center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although copper ions are essential for the activity of tyrosinase, the mechanism of copper uptake is still unclear. We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison with CuB. Investigating copper accumulation in TyrBm, we found that the presence of copper has a more significant effect on the diphenolase activity. By decreasing the concentration of copper, we increased the diphenolase to monophenolase activity ratio twofold. Using a rational design approach, we identified five variants having an impact on copper uptake. We have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Further investigation of these variants revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, not only play a role in copper uptake, but are also important for enhancing the diphenolase activity. We propose a mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards L-dopa production.

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 Å resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended α-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.

Solar-to-hydrogen generation is a promising approach to generate clean and renewable fuel. Nanohybrid structures such as CdSe@CdS-Pt nanorods were found favorable for this task (attaining 100% photon-to-hydrogen production efficiency); yet the rods cannot support overall water splitting. The key limitation seems to be the rate of hole extraction from the semiconductor, jeopardizing both activity and stability. It is suggested that hole extraction might be improved via tuning the rod’s dimensions, specifically the width of the CdS shell around the CdSe seed in which the holes reside. In this contribution, we successfully attain atomic-scale control over the width of CdSe@CdS nanorods, which enables us to verify this hypothesis and explore the intricate influence of shell diameter over hole quenching and photocatalytic activity towards H2 production. A non-monotonic effect of the rod’s diameter is revealed, and the underlying mechanism for this observation is discussed, alongside implications towards the future design of nanoscale photocatalysts.

The conversion of solar energy (SEC) to storable chemical energy by photosynthesis has been performed by photosynthetic organisms, including oxygenic cyanobacteria for over 3 billion years. We have previously shown that crude thylakoid membranes from the cyanobacterium Synechocytis sp. PCC 6803 can reduce the electron transfer (ET) protein cytochrome c even in the presence of the PSII inhibitor DCMU. Mutation of lysine 238 of the Photosystem II D1 protein to glutamic acid increased the cytochrome reduction rates, indicating the possible position of this unknown ET pathway. In this contribution, we show that D1-K238E is rather unique, as other mutations to K238, or to other residues in the same vicinity, are not as successful in cytochrome c reduction. This observation indicates the sensitivity of ET reactions to minor changes. As the next step in obtaining useful SEC from biological material, we describe the use of crude Synechocystis membranes in a bio-photovoltaic cell containing an N-acetyl cysteine-modified gold electrode. We show the production of significant current for prolonged time durations, in the presence of DCMU. Surprisingly, the presence of cytochrome c was not found to be necessary for ET to the bio-voltaic cell.

Reverse transcriptases (RTs) exhibit DNA polymerase and ribonuclease H (RNase H) activities. The RTs of human immunodeficiency viruses type 1 and type 2 (HIV‐1 and HIV‐2) are composed of two subunits, both sharing the same N‐terminus (which encompasses the DNA polymerase domain). The smaller subunit lacks the C‐terminal segment of the larger one, which contains the RNase H domain. The DNA polymerase domain of RTs resembles a right hand linked to the RNase H domain by a connection subdomain. Despite the high homology between HIV‐1 and HIV‐2 RTs, the RNase H activity of the latter is substantially lower than that of HIV‐1 RT. The thumb subdomain of the small subunit controls the level of RNase H activity. We show here that Gln294, located in this thumb, is responsible for this difference in activity. A HIV‐2 RT mutant, where Gln294 in the small subunit was replaced by a proline (present in HIV‐1 RT), has an activity almost 10‐fold higher than that of the wild‐type RT. A comparative in vitro study of the kinetic parameters of the RNase H activity suggests that residue 294 affects the Km rather than the kcat value, influencing the affinity for the RNA·DNA substrate.

Light absorption is the initial step in the photosynthetic process. In all species, most of the light is absorbed by dedicated pigment-protein complexes called light harvesting complexes or antenna complexes. In the case of cyanobacteria and red-algae, photosynthetic organisms found in a wide variety of ecological niches, the major antenna is called the Phycobilisome (PBS). The PBS has many unique characteristics that sets it apart from the antenna complexes of other organisms (bacteria, algae and plants). These differences include the type of light absorbing chromophores, the protein environment of the chromophores, the method of assembly and association and the intercellular location with respect to the photosynthetic reaction centers (RCs). Since the final goal of all antenna complexes is the same – controlled absorption and transfer of the energy of the sun to the RCs, the unique structural and chemical differences of the PBS also require unique energy transfer mechanisms and pathways. In this review we will describe in detail the structural facets that lead to a mature PBS, followed by an attempt to understand the energy transfer properties of the PBS as they have been measured experimentally.

Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor.

Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein–protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.

Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically, meiosis occurs in MATa/MATα cells shifted to nitrogen depletion media in the absence of glucose and the presence of a nonfermentable carbon source. These conditions lead to the expression and activation of Ime 1, the master regulator of meiosis. IME1 encodes a transcriptional activator recruited to promoters of early meiosis-specific genes by association with the DNA-binding protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6. Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of the early genes promote entry into the meiotic cycle, as they include genes required for premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination. Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homologue, Ime2, are required for the transcription of middle meiosis-specific genes that are involved with nuclear division and spore formation. Spore maturation depends on late genes whose expression is indirectly dependent on Ime1, Ime2, and Ndt80. Finally, phosphorylation of Ime1 by Ime2 leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade.

The increase in world energy consumption, and the worries from potential future disasters that may derive from climate change have stimulated the development of renewable energy technologies. One promising method is the utilization of whole photosynthetic cyanobacterial cells to produce photocurrent in a bio-photo electrochemical cell (BPEC). The photocurrent can be derived from either the respiratory or photosynthetic pathways, via the redox couple NADP+/NADPH mediating cyclic electron transport between photosystem I inside the cells, and the anode. In the past, most studies have utilized the fresh-water cyanobacterium Synechocystis sp. PCC 6803 (Syn). Here, we show that the globally important marine cyanobacterium Trichodesmium erythraeum flourishing in the subtropical oceans can provide improved currents as compared to Syn. We applied 2D-fluorescence measurements to detect the secretion of NADPH and show that the resulting photocurrent production is enhanced by increasing the electrolyte salinity, Further enhancement of the photocurrent can be obtained by the addition of electron mediators such as NAD+, NADP+, cytochrome C, vitamin B1, or potassium ferricyanide. Finally, we produce photocurrent from additional cyanobacterial species: Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, Acaryochloris marina MBIC 11017, and Spirulina, using their cultivation media as electrolytes for the BPEC.

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.

Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry. The disease is occasionally observed to affect women in families in a manner that suggests an autosomal dominant trait and usually manifests clinically after intense hair grooming. We sought to determine whether there exists a genetic basis of CCCA and, if so, what it is.

Cyanobacteria of the genera Synechococcus and Prochlorococcus are important contributors to photosynthetic productivity in the open ocean. The discovery of genes (psbA, psbD) that encode key photosystem II proteins (D1, D2) in the genomes of phages that infect these cyanobacteria suggests new paradigms for the regulation, function and evolution of photosynthesis in the vast pelagic ecosystem. Reports on the prevalence and expression of phage photosynthesis genes, and evolutionary data showing a potential recombination of phage and host genes, suggest a model in which phage photosynthesis genes help support photosynthetic activity in their hosts during the infection process. Here, using metagenomic data in natural ocean samples, we show that about 60% of the psbA genes in surface water along the global ocean sampling transect are of phage origin, and that the phage genes are undergoing an independent selection for distinct D1 proteins. Furthermore, we show that different viral psbA genes are expressed in the environment.

Attachment of bilins to phycobiliproteins is performed by dedicated lyases. In this issue of Structure, Kumarapperuma et al., 2022 present the structure of an E/F type lyase-isomerase that identifies the correct biological interface between active domains, suggesting that a previous E/F lyase misidentified the heterodimer structure from the crystal lattice.