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.

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.

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.

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.

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.

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.

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 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 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.

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.

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.

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 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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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 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

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.

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.

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.

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.’

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.