Global spatial risk assessment of sharks under the footprint of fisheries.

Effective ocean management and the conservation of highly migratory species depend on resolving the overlap between animal movements and distributions, and fishing effort. However, this information is lacking at a global scale. Here we show, using a big-data approach that combines satellite-tracked movements of pelagic sharks and global fishing fleets, that 24% of the mean monthly space used by sharks falls under the footprint of pelagic longline fisheries. Space-use hotspots of commercially valuable sharks and of internationally protected species had the highest overlap with longlines (up to 76% and 64%, respectively), and were also associated with significant increases in fishing effort. We conclude that pelagic sharks have limited spatial refuge from current levels of fishing effort in marine areas beyond national jurisdictions (the high seas). Our results demonstrate an urgent need for conservation and management measures at high-seas hotspots of shark space use, and highlight the potential of simultaneous satellite surveillance of megafauna and fishers as a tool for near-real-time, dynamic management.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Alternatives to Styrene- and Diisobutylene-Based Copolymers for Membrane Protein Solubilization via Nanodisc Formation.

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.

Effects of Esterified Styrene-Maleic Acid Copolymer Degradation on Integral Membrane Protein Extraction.

The detergent-free extraction of integral membrane proteins using styrene-maleic acid copolymers (SMAs) has shown promise as a potentially effective technique to isolate proteins in a more native-like conformation. As the field continues to develop, the protein selectivity and extraction efficiency of many analogues of traditional SMAs are being investigated. Recently, we discovered that the monoesterification of SMAs with alkoxy ethoxylate sidechains drastically affects the bioactivity of these copolymers in the extraction of photosystem I from the cyanobacterium Thermosynechococcus elongatus. However, subsequent investigations also revealed that the conditions under which these esterified SMA polymer analogues are prepared, purified, and stored can alter the structure of the alkoxy ethoxylate-functionalized SMA and perturb the protein extraction process. Herein, we demonstrate that the basic conditions required to solubilize SMA analogues may lead to deleterious saponification side reactions, cleaving the sidechains of an esterified SMA and dramatically decreasing its efficacy for protein extraction. We found that this process is highly dependent on temperature, with polymer samples being prepared and stored at lower temperatures exhibiting significantly fewer saponification side reactions. Furthermore, the effects of small-molecule impurities and exposure to light were also investigated, both of which are shown to have significant effects on the polymer structure and/or protein extraction process.

PEDOT-Carbon Nanotube Counter Electrodes and Bipyridine Cobalt (II/III) Mediators as Universally Compatible Components in Bio-Sensitized Solar Cells Using Photosystem I and Bacteriorhodopsin.

In nature, solar energy is captured by different types of light harvesting protein-pigment complexes. Two of these photoactivatable proteins are bacteriorhodopsin (bR), which utilizes a retinal moiety to function as a proton pump, and photosystem I (PSI), which uses a chlorophyll antenna to catalyze unidirectional electron transfer. Both PSI and bR are well characterized biochemically and have been integrated into solar photovoltaic (PV) devices built from sustainable materials. Both PSI and bR are some of the best performing photosensitizers in the bio-sensitized PV field, yet relatively little attention has been devoted to the development of more sustainable, biocompatible alternative counter electrodes and electrolytes for bio-sensitized solar cells. Careful selection of the electrolyte and counter electrode components is critical to designing bio-sensitized solar cells with more sustainable materials and improved device performance. This work explores the use of poly (3,4-ethylenedioxythiophene) (PEDOT) modified with multi-walled carbon nanotubes (PEDOT/CNT) as counter electrodes and aqueous-soluble bipyridine cobaltII/III complexes as direct redox mediators for both PSI and bR devices. We report a unique counter electrode and redox mediator system that can perform remarkably well for both bio-photosensitizers that have independently evolved over millions of years. The compatibility of disparate proteins with common mediators and counter electrodes may further the improvement of bio-sensitized PV design in a way that is more universally biocompatible for device outputs and longevity.

Protein Extraction Efficiency and Selectivity of Esterified Styrene-Maleic Acid Copolymers in Thylakoid Membranes.

Amphiphilic styrene-maleic acid copolymers (SMAs) have been shown to effectively extract membrane proteins surrounded by an annulus of native membrane lipids via the formation of nanodiscs. Recent reports have shown that 2-butoxyethanol-functionalized SMA derivatives promote the extraction of membrane proteins from thylakoid membranes, whereas unfunctionalized SMA is essentially ineffective. However, it is unknown how the extent of functionalization and identity of sidechains impact protein solubilization and specificity. Herein, we show that the monoesterification of an SMA polymer with hydrophobic alkoxy ethoxylate sidechains leads to an increased solubilization efficiency (SE) of trimeric photosystem I (PSI) from the membranes of cyanobacterium Thermosynechococcus elongatus. The specific SMA polymer used in this study, PRO 10235, cannot encapsulate single PSI trimers from this cyanobacterium; however, as it is functionalized with alkoxy ethoxylates of increasing alkoxy chain length, a clear increase in the trimeric PSI SE is observed. Furthermore, an exponential increase in the SE is observed when >50% of the maleic acid repeat units are monoesterified with long alkoxy ethoxylates, suggesting that the PSI extraction mechanism is highly dependent on both the number and length of the attached side chains.

PSI-SMALP, a Detergent-free Cyanobacterial Photosystem I, Reveals Faster Femtosecond Photochemistry.

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c6-ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-ß-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.1-20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700+A1- occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the long-wavelength chlorophyll to P700. Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultrafast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%), the energy transfer to P700 occurred at ∼36 ps, similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation.

X-ray and Neutron Reflectivity Studies of Styrene-Maleic Acid Copolymer Interactions with Galactolipid-Containing Monolayers.

Styrene-maleic acid (SMA) copolymers have recently gained attention for their ability to facilitate the detergent-free solubilization of membrane protein complexes and their native boundary lipids into polymer-encapsulated, nanosized lipid particles, referred to as SMALPs. However, the interfacial interactions between SMA and lipids, which dictate the mechanism, efficiency, and selectivity of lipid and membrane protein extraction, are barely understood. Our recent finding has shown that SMA 1440, a chemical derivative of the SMA family with a functionalized butoxyethanol group, was most active in galactolipid-rich membranes, as opposed to phospholipid membranes. In the present work, we have performed X-ray reflectometry (XRR) and neutron reflectometry (NR) on the lipid monolayers at the liquid-air interface followed by the SMA copolymer adsorption. XRR and Langmuir Π-A isotherms captured the fluidifying effect of galactolipids, which allowed SMA copolymers to infiltrate easily into the lipid membranes. NR results revealed the detailed structural arrangement of SMA 1440 copolymers within the membranes and highlighted the partition of butoxyethanol group into the lipid tail region. This work allows us to propose a possible mechanism for the membrane solubilization by SMA.

Cryo-EM structure of a tetrameric cyanobacterial photosystem I complex reveals novel subunit interactions.

Photosystem I (PSI) of the thermophilic cyanobacterium Chroococcidiopsis sp. TS-821 (TS-821) forms tetramers Li et al. (2014). Two-dimensional maps obtained by single particle electron microscopy (EM) clearly show that the tetramer lacks four-fold symmetry and is actually composed of a dimer of dimers with C2 symmetry. The resolution of these negative stain 2D maps did not permit the placement of most of the small PSI subunits, except for PsaL. Therefore cryo-EM was used for 3D reconstruction of the PSI tetramer complex. A 3D model at ~11.5Å resolution was obtained and a 2D map within the membrane plane of ~6.1Å. This data was used to build a model that was compared with the high-resolution structure of the PSI of Thermosynechococcus elongatus (T. elongatus) at 2.5Å. This comparison reveals key differences in which subunits are involved in the two different interfaces, interface type 1 within a dimer and interface type 2 between dimers. The type 1 interface in TS-821 is similar to the monomer interface in the trimeric PSI from T. elongatus, with interactions between subunits PsaA, -B, -I, -L and M. In type 2 the interaction is only between PsaA, -B and -L. Unlike the trimeric PSI, the central cavity of the complex is not filled with the PsaL-derived helical bundle, but instead seems filled with lipids. The physiological or evolutionary advantage of the tetramer is unknown. However, the presence of both dimers and tetramers in the thylakoid membrane suggest a dynamic equilibrium that shifts towards the tetramers in high light.

In vitro kinetics of P700+ reduction of Thermosynechococcus elongatus trimeric Photosystem I complexes by recombinant cytochrome c 6 using a Joliot-type LED spectrophotometer.

The reduction rate of photo-oxidized Photosystem I (PSI) with various natural and artificial electron donors have been well studied by transient absorption spectroscopy. The electron transfer rate from various donors to P700+ has been measured for a wide range of photosynthetic organisms encompassing cyanobacteria, algae, and plants. PSI can be a limiting component due to tedious extraction and purification methods required for this membrane protein. In this report, we have determined the in vivo, intracellular cytochrome c 6 (cyt c 6)/PSI ratio in Thermosynechococcus elongatus (T.e.) using quantitative Western blot analysis. This information permitted the determination of P700+ reduction kinetics via recombinant cyt c 6 in a physiologically relevant ratio (cyt c 6: PSI) with a Joliot-type, LED-driven, pump-probe spectrophotometer. Dilute PSI samples were tested under varying cyt c 6 concentration, temperature, pH, and ionic strength, each of which shows similar trends to the reported literature utilizing much higher PSI concentrations with laser-based spectrophotometer. Our results do however indicate kinetic differences between actinic light sources (laser vs. LED), and we have attempted to resolve these effects by varying our LED light intensity and duration. The standardized configuration of this spectrophotometer will also allow a more uniform kinetic analysis of samples in different laboratories. We can conclude that our findings from the LED-based system display an added total protein concentration effect due to multiple turnover events of P700+ reduction by cyt c 6 during the longer illumination regime.

Characterization and evolution of tetrameric photosystem I from the thermophilic cyanobacterium Chroococcidiopsis sp TS-821.

Photosystem I (PSI) is a reaction center associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in green and purple bacteria, PSI forms trimeric complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a tetrameric form of PSI in the thermophilic cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821, PSI forms tetrameric and dimeric species. We investigated these species by Blue Native PAGE, Suc density gradient centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis. Transmission electron microscopy analysis of native membranes confirms the presence of the tetrameric PSI structure prior to detergent solubilization. To investigate why TS-821 forms tetramers instead of trimers, we cloned and analyzed its psaL gene. Interestingly, this gene product contains a short insert between the second and third predicted transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which also have a tetrameric form of PSI. These results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to tetrameric oligomer en route to becoming monomeric in plants/algae.

Non-native, N-terminal Hsp70 molecular motor recognition elements in transit peptides support plastid protein translocation.

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences.

Enhanced photocurrent from Photosystem I upon in vitro truncation of the antennae chlorophyll.

Current effects on climate change and dwindling fossil fuel reserves require new materials and methods to convert solar energy into a viable clean energy source. Recent progress in the direct conversion of light into photocurrent has been well documented using Photosystem I. In plants, PSI consists of a core complex and multiple light-harvesting complexes, denoted LHCI and LHCII. Most of the methods for isolating PSI from plants involve a selective, detergent solubilization from thylakoids followed by sucrose gradient density centrifugation. These processes isolate one variant of PSI with a specific ratio of Chl:P700. In this study, we have developed a simple and potentially scalable method for isolating multiple PSI variants using Hydroxyapatite chromatography, which has been well documented in other Photosystem I isolation protocols. By varying the wash conditions, we show that it is possible to change the Chl:P700 ratios. These different PSI complexes were cast into a PSI-Nafion-osmium polymer film that enabled their photoactivity to be measured. Photocurrent increases nearly 400% between highly washed and untreated solutions based on equal chlorophyll content. Importantly, the mild washing conditions remove peripheral Chl and some LHCI without inhibiting the photochemical activity of PSI as suggested by SDS-PAGE analysis. This result could indicate that more P700 could be loaded per surface area for biohybrid devices. Compared with other PSI isolations, this protocol also allows isolation of multiple PSI variants without loss of photochemical activity.

Correction to Thermodynamic Characterization of a Thermostable Antibiotic Resistance Enzyme, the Aminoglycoside Nucleotidyltransferase (4').

Biochemistry 2012, 51 (45), 9147−9155. DOI: 10.1021/bi301126g. Page 9148. A corrected version of the Figure 2 legend appears here: Figure 2. Backbone of the ANT D80Y variant in ribbon representation. Two monomer subunits are colored red and green. Bound kanamycin A molecules are colored blue, and Mg-AMPCPP molecules are colored yellow (Protein Data Bank entry 1KNY).14 Page 9148 (last line). The sentence should read, "A thermostable variant of ANT, T130K, was obtained from thermophilic cyanobacterium T. elongatus."

Growing green electricity: progress and strategies for use of photosystem I for sustainable photovoltaic energy conversion.

Oxygenic photosynthesis is driven via sequential action of Photosystem II (PSII) and (PSI)reaction centers via the Z-scheme. Both of these pigment-membrane protein complexes are found in cyanobacteria, algae, and plants. Unlike PSII, PSI is remarkably stable and does not undergo limiting photo-damage. This stability, as well as other fundamental structural differences, makes PSI the most attractive reaction centers for applied photosynthetic applications. These applied applications exploit the efficient light harvesting and high quantum yield of PSI where the isolated PSI particles are redeployed providing electrons directly as a photocurrent or, via a coupled catalyst to yield H2. Recent advances in molecular genetics, synthetic biology, and nanotechnology have merged to allow PSI to be integrated into a myriad of biohybrid devices. In photocurrent producing devices, PSI has been immobilized onto various electrode substrates with a continuously evolving toolkit of strategies and novel reagents. However, these innovative yet highly variable designs make it difficult to identify the rate-limiting steps and/or components that function as bottlenecks in PSI-biohybrid devices. In this study we aim to highlight these recent advances with a focus on identifying the similarities and differences in electrode surfaces, immobilization/orientation strategies, and artificial redox mediators. Collectively this work has been able to maintain an annual increase in photocurrent density (Acm⁻²) of ~10-fold over the past decade. The potential drawbacks and attractive features of some of these schemes are also discussed with their feasibility on a large-scale. As an environmentally benign and renewable resource, PSI may provide a new sustainable source of bioenergy. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Engineering Photosystem I Complexes with Metal Oxide Binding Peptides for Bioelectronic Applications.

Conventional dye-sensitized solar cells comprise semiconducting anodes sensitized with complex synthetic organometallic dyes, a platinum counter electrode, and a liquid electrolyte. This work focuses on replacing synthetic dyes with a naturally occurring biological pigment-protein complex known as Photosystem I (PSI). Specifically, ZnO binding peptides (ZOBiP)-fused PSI subunits (ZOBiP-PsaD and ZOBiP-PsaE) and TiO2 binding peptides (TOBiP)-fused ferredoxin (TOBiP-Fd) have been produced recombinantly from Escherichia coli. The MOBiP-fused peptides have been characterized via western blotting, circular dichroism, MALDI-TOF, and cyclic voltammetry. ZOBiP-PSI subunits have been used to replace wild-type PsaD and PsaE, and TOBiP-Fd has been chemically cross-linked to the stromal hump of PSI. These MOBiP peptides and MOBiP-PSI complexes have been produced and incubated with various metal oxide nanoparticles, showing increased binding when compared to that of wild-type PSI complexes.

Differential transit peptide recognition during preprotein binding and translocation into flowering plant plastids.

Despite the availability of thousands of transit peptide (TP) primary sequences, the structural and/or physicochemical properties that determine TP recognition by components of the chloroplast translocon are not well understood. By combining a series of in vitro and in vivo experiments, we reveal that TP recognition is determined by sequence-independent interactions and vectorial-specific recognition domains. Using both native and reversed TPs for two well-studied precursors, small subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase, and ferredoxin, we exposed these two modes of recognition. Toc34 receptor (34-kD subunit of the translocon of the outer envelope) recognition in vitro, preprotein binding in organellar, precursor binding in vivo, and the recognition of TPs by the major stromal molecular motor Hsp70 are specific for the physicochemical properties of the TP. However, translocation in organellar and in vivo demonstrates strong specificity to recognition domain organization. This organization specificity correlates with the N-terminal placement of a strong Hsp70 recognition element. These results are discussed in light of how individual translocon components sequentially interact with the precursor during binding and translocation and helps explain the apparent lack of sequence conservation in chloroplast TPs.

Molecular interactions between photosystem I and ferredoxin: an integrated energy frustration and experimental model.

The stromal domain (PsaC, PsaD, and PsaE) of photosystem I (PSI) reduces transiently bound ferredoxin (Fd) or flavodoxin. Experimental structures exist for all of these protein partners individually, but no experimental structure of the PSI/Fd or PSI/flavodoxin complexes is presently available. Molecular models of Fd docked onto the stromal domain of the cyanobacterial PSI site are constructed here utilizing X-ray and NMR structures of PSI and Fd, respectively. Predictions of potential protein-protein interaction regions are based on experimental site-directed mutagenesis and cross-linking studies to guide rigid body docking calculations of Fd into PSI, complemented by energy landscape theory to bring together regions of high energetic frustration on each of the interacting proteins. The results identify two regions of high localized frustration on the surface of Fd that contain negatively charged Asp and Glu residues. This study predicts that these regions interact predominantly with regions of high localized frustration on the PsaC, PsaD, and PsaE chains of PSI, which include several residues predicted by previous experimental studies.

Analysis of the solution structure of Thermosynechococcus elongatus photosystem I in n-dodecyl-ß-D-maltoside using small-angle neutron scattering and molecular dynamics simulation.

Small-angle neutron scattering (SANS) and molecular dynamics (MD) simulation were used to investigate the structure of trimeric photosystem I (PSI) from Thermosynechococcus elongatus (T. elongatus) stabilized in n-dodecyl-ß-d-maltoside (DDM) detergent solution. Scattering curves of detergent and protein-detergent complexes were measured at 18% D2O, the contrast match point for the detergent, and 100% D2O, allowing observation of the structures of protein/detergent complexes. It was determined that the maximum dimension of the PSI-DDM complex was consistent with the presence of a monolayer belt of detergent around the periphery of PSI. A dummy-atom reconstruction of the shape of the complex from the SANS data indicates that the detergent envelope has an irregular shape around the hydrophobic periphery of the PSI trimer rather than a uniform, toroidal belt around the complex. A 50 ns MD simulation model (a DDM ring surrounding the PSI complex with extra interstitial DDM) of the PSI-DDM complex was developed for comparison with the SANS data. The results suggest that DDM undergoes additional structuring around the membrane-spanning surface of the complex instead of a simple, relatively uniform belt, as is generally assumed for studies that use detergents to solubilize membrane proteins.