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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.

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.

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–Aisotherms 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.

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 elongatusvia conventional n-dodecyl-β-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 P700occurred 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.

Photosystem I (PSI) is present as trimeric complexes in most characterized cyanobacteria and as monomers in plants and algae. Recent reports of tetrameric PSI have raised questions regarding its structural basis, physiological role, phylogenetic distribution and evolutionary significance. Here, we examined PSI in 61 cyanobacteria, showing that tetrameric PSI, which correlates with the psaLgene and a distinct genomic structure, is widespread among heterocyst-forming cyanobacteria and their close relatives. Physiological studies revealed that expression of tetrameric PSI is favoured under high light, with an increased content of novel PSI-bound carotenoids (myxoxanthophyll, canthaxanthan and echinenone). In sum, this work suggests that tetrameric PSI is an adaptation to high light intensity, and that change in PsaL leads to monomerization of trimeric PSI, supporting the hypothesis of tetrameric PSI being the evolutionary intermediate in the transition from cyanobacterial trimeric PSI to monomeric PSI in plants and algae.

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.

Morphological variations of Photosystem I (PS I) assembly on hydroxyl-terminated alkanethiolate self-assembled monolayer (SAM)/Au substrates with various deposition techniques is presented. Our studies indicate that deposition conditions such as PS I concentration and driving force play a central role in determining organization of immobilized PS I on thiol-activated Au surfaces. Specifically, atomic force microscopy (AFM) and ellipsometry analyses indicate that gravity-driven deposition from concentrated PS I solutions results in a large number of columnar PS I aggregates, which assemble perpendicular to the Au surface. PS I deposition yields much more uniform layers when deposited at lower concentrations, suggesting preassembly of the aggregate formation in the solution phase. Moreover, in electric-field assisted deposition at high field strengths, columnar self-assembly is largely prevented, thereby allowing a uniform, monolayer-like deposition even at very high PS I concentrations. In situ dynamic light scattering (DLS) studies of solution-phase aggregation dynamics of PS I suspensions in both the presence and absence of an applied electric field support these observations and clearly demonstrate that the externally imposed electric field effectively fragments large PS I aggregates in the solution phase, thereby permitting a uniform deposition of PS I trimers on SAM/Au substrates.

The antimicrobial activity of eugenol microemulsions (eugenol encapsulated in surfactant micelles) in ultrahigh-temperature pasteurized milk containing different percentages of milk fat (0, 2, and 4%) was investigated. Antimicrobial micro-emulsions were prepared from a 5% (wt) aqueous surfactant solution (Surfynol 485W) with 0.5% (wt) eugenol. Two strains each of Listeria monocytogenesand Escherichia coliO157:H7 previously shown to be the least and most resistant to the microemulsion in microbiological media were used to inoculate sterile milk (104CFU/ml). Samples were withdrawn and plated at 0, 1, 3, 6, 12, and 24 h for enumeration. Microemulsions completely prevented growth of L. monocytogenesfor up to 48 h in skim milk and reduced both strains of E. coliO157:H7 to less than detectable levels in less than 1 h. Similarly, in 2% fat milk, eugenol-Surfynol combinations reduced both strains of E. coliO157:H7 to less than detectable levels in less than 1 h but only increased the lag phase of both strains of L. monocytogenes. In full-fat milk (4% fat), microemulsions inhibited growth of the least resistant strains of L. monocytogenesand E. colibut were ineffective against the two resistant strains. Unencapsulated eugenol was slightly more or as inhibitory as microemulsions against target pathogens. Results were attributed to diffusional mass transport of antimicrobials from microemulsions to the macroemulsion (milk). Results suggest that food composition, especially fat level, may affect the efficiency of targeting of foodborne pathogens with surfactant-encapsulated antimicrobials.

Growth inhibition of four strains of Escherichia coliO157:H7 (H1730, F4546, 932, and E0019) and Listeria monocytogenes(Scott A, 101, 108, and 310) by essential oil components (carvacrol and eugenol) solubilized in nonionic surfactant micelles (Surfynol 465 and 485W) was investigated. Concentrations of encapsulated essential oil components ranged from 0.02 to 1.25% depending on compound, surfactant type, and surfactant concentration (0.5 to 5%). Eugenol encapsulated in Surfynol 485W micelles was most efficient in inhibiting growth of the pathogens; 1% Surfynol 485W and 0.15% eugenol was sufficient to inhibit growth of all strains of E. coliO157:H7 and three of four strains of L. monocytogenes(Scott A, 310, and 108). The fourth strain, L. monocytogenes101, was inhibited by 2.5% Surfynol and 0.225% eugenol. One percent Surfynol 485W in combination with 0.025% carvacrol was effective in inhibiting three of four strains of E. coliO157:H7. Strain H1730 was the most resistant strain, requiring 0.3% carvacrol and 5% surfactant for complete inhibition. Growth inhibition of L. monocytogenesby combinations of carvacrol and Surfynol 465 ranged between 0.15 and 0.35% and 1 and 3.75%, respectively. Generally, the antimicrobial activity of Surfynol 465 in combination with eugenol was higher than that for the combination with carvacrol. The potent activity was attributed to increased solubility of essential oil components in the aqueous phase due to the presence of surfactants and improved interactions of antimicrobials with microorganisms.

Growth inhibition of four strains of Escherichia coliO157:H7 (H1730, F4546, 932, and E0019) and Listeria monocytogenes(Scott A, 101, 108, and 310) by eugenol encapsulated in water soluble micellar nonionic surfactant solutions (Surfynol 485W) adjusted to pH 5, 6, and 7 and incubated at 10, 22, and 32°C was determined. Concentrations of eugenol ranged from 0.2 to 0.9% at a surfactant concentration of 5%. Antimicrobial activity was assessed using a microbroth dilution assay. Eugenol encapsulated in surfactant micelles inhibited both microorganisms at pH 5, 6, and 7. At pH 5, some inhibition occurred in the absence of eugenol, i.e., by the surfactant itself (optical density at 24 h for L. monocytogenes= 0.07 and optical density at 24 h for E. coliO157:H7 = 0.09), but addition of >0.2% eugenol led to complete inhibition of both microorganisms. Inhibition of L. monocytogenesand E. coliO157:H7 decreased with increasing pH, that is, the minimum inhibitory concentration was 0.2, 0.5, and 0.5% of micellar encapsulated eugenol solutions at pH 5, 6, and 7, respectively. The encapsulated essential oil component in surfactant micelles was effective at all three temperatures tested (10, 22, and 32°C), indicating that the activity of encapsulated eugenol was not affected by high or low (refrigeration) temperatures. Overall, strains of E. coliO157:H7 were more sensitive than strains of L. monocytogenes. Improved activity was attributed to increased solubility of eugenol in the aqueous phase due to the presence of surfactants and improved interactions of antimicrobials with microorganisms.

ABSTRACTHeat shock proteins loaded with viral peptides were shown to induce a CD8+T cell response and confer protective immunity against challenge with herpes simplex virus (HSV). The delivery system consisted of recombinant human hsp70 coupled to the peptide SSIEFARL, which is the immunodominant peptide epitope, recognized by HSV specific T cells in C57BL/6 mice. Immunization resulted in CD8+T-cell responses, measured by peptide-specific tetramers and peptide-induced intracellular gamma interferon expression and cytotoxicity, similar to responses resulting from immunization with a recombinant vaccinia virus that expressed SSIEFARL as a minigene (VvgB) and UV-inactivated HSV. However, the durability of the hsp70-SSIEFARL response was less than that resulting from VvgB and HSV immunization and in addition the CD8+T-cell responses in the memory phase were functionally less effective. Mice challenged soon after immunization showed excellent immunity, but by 90 days postimmunization this had waned to be significantly less than the level of immunity in both VvgB- and HSV-immunized mice.

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T= 3 icosahedral symmetry. The particle is composed of 180 subunits of the coat protein (CP) and one copy of the positive-sense viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 aa and the shell (S) domain (aa 65–260). The R domain is highly charged, with 11 of the N-terminal 30 residues being basic. It is localized to the interior of the native particle where it may interact with the viral RNA, but under certain pH and salt conditions the topology of the particle changes to externalize the R domain. Since the CPs of several spherical RNA viruses have been shown to interact with host membranes during infection, we have begun investigating the membrane interactions of the SCPMV CP using the artificial liposome membranes. Both the native CP and the R domain overexpressed in Escherichia coliwere observed to interact with liposomes. The interaction between the R domain and liposomes required either anionic phospholipids or non-bilayer-forming lipids and involved electrostatic interactions since it was shown to be both pH and ionic strength dependent. The analysis of four different deletion and six different site-directed substitution mutations partially mapped the region responsible for this interaction to residues 1–30. Analysis of this region of the R domain by circular dichroism indicated that it assumes an α-helical structure when exposed to liposomes composed of anionic lipids. Mutations, which extend the helical nature of this region, promoted an increased interaction. The possible role of the CP/lipid interaction in the SCPMV infection is discussed.

Small numbers of blue grenadier, Macruronus novaezelandiae, larvae were found in coastal waters off eastern Victoria and southern New South Wales in August 1993. This is the first record of larval blue grenadier from mainland Australian waters. It is considerably further north than previous records of larvae and remote from the single known spawning ground off western Tasmania. Larvae were aged between 17 and 36 days and were largely confined to an inshore northward flowing water mass. Back calculated spawning dates indicated that larvae from eastern Victoria/southern NSW were spawned earlier than larvae collected during the same period off western and southern Tasmania. Otolith increment widths were significantly wider in larvae caught in eastern Victoria/southern NSW suggesting that they experienced faster growth and development conditions than the Tasmanian larvae. Three-dimensional modelling of circulation and particle advection suggested that the source of eastern Victoria/southern NSW larvae was most likely eastern Bass Strait. These data suggest that there is a second, albeit limited, spawning area for blue grenadier in south-eastern Australia.

In Arabidopsisseedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In ArabidopsisCOP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

The mechanism of import-competent precursor protein-induced inactivation of a 50-pS anion channel of the chloroplast envelope is investigated using single-channel recordings. The inactivation by precursor protein is the result of the induction of a long-lived closed state of the channel. The mean duration of this state does not depend on precursor concentration. From this it can be concluded that the protein import related anion channel enters the inactive state less frequently when the precursor concentration is lowered, but that the time spent in this state remains the same. Furthermore, it was found that the presence of precursor protein also decreases the mean durations of preexisting open and closed states of the channel. This decrease is found to be dependent on the precursor concentration. From this it is concluded that there is a direct interaction between the precursor protein and a protein complex of which the channel is a constituent. The mean duration of the precursor-induced long-lived closed state does not depend on the length of the translocation-competent precursor. This suggests that the duration of import is independent of precursor length.

The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Δ52, Δ67, and Δ74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of 35S-prSSU. Both IC50and Kivalues consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Δ52 were properly processed and accumulated within the chloroplast, whereas Δ67 and Δ74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.

Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, chloroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins for their ability to immune-isolate chloroplasts.

Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron microscopy. Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that lipid interaction is mediated primarily through the transit peptide. Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release. Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms. Interaction between the peptides and bilayer was also lipid class-dependent. Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids. These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer HIIstructures.

Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, ß-globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, ß-globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.

Photochemical activity was examined in membrane fragments and a purified membrane preparation from Chloroflexus. Flash-induced absorption difference spectroscopy strongly suggests a primary donor (P865) that is more similar to the P870bacteriochlorophyll adimer found in the purple photosynthetic bacteria than it is to P840found in the anaerobic green bacteria. Redox measurements on P865and an early acceptor also indicate a photochemical system characteristic of the purple bacteria. The membrane preparation contains a tightly bound type ccytochrome, c554, that is closely coupled to the reaction center as indicated by its ability to rereduce photooxidized P865. Chloroflexusthus appears to be distinct photochemically from other families of photosynthetic bacteria and may occupy an important role in photosynthetic evolution.

Photosystem I (PSI) is one of two the photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, which is in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutional significance. Here we report the ∼3.72 Å resolution cryo-EM structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsissp. TS-821. The structure resolves 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.