Methods for Interpreting the Partitioning and Fate of Petroleum Hydrocarbons in a Sea Ice Environment.

Decreases in Arctic Sea ice extent and thickness have led to more open ice conditions, encouraging both shipping traffic and oil exploration within the northern Arctic. As a result, the increased potential for accidental releases of crude oil or fuel into the Arctic environment threatens the pristine marine environment, its ecosystem, and local inhabitants. Thus, there is a need to develop a better understanding of oil behavior in a sea ice environment on a microscopic level. Computational quantum chemistry was used to simulate the effects of evaporation, dissolution, and partitioning within sea ice. Vapor pressures, solubilities, octanol-water partition coefficients, and molecular volumes were calculated using quantum chemistry and thermodynamics for pure liquid solutes (oil constituents) of interest. These calculations incorporated experimentally measured temperatures and salinities taken throughout an oil-in-ice mesocosm experiment conducted at the University of Manitoba in 2017. Their potential for interpreting the relative movements of oil constituents was assessed. Our results suggest that the relative movement of oil constituents is influenced by differences in physical properties. Lighter molecules showed a greater tendency to be controlled by brine advection processes due to their greater solubility. Molecules which are more hydrophobic were found to concentrate in areas of lower salt concentration.

Investigation into the geometry and distribution of oil inclusions in sea ice using non-destructive X-ray microtomography and its implications for remote sensing and mitigation potential.

As climate change brings reduced sea ice cover and longer ice-free summers to the Arctic, northern Canada is experiencing an increase in shipping and industrial activity in this sensitive region. Disappearing sea ice, therefore, makes the Arctic region susceptible to accidental releases of different types of oil and fuel pollution resulting in a pressing need for the development of appropriate scientific knowledge necessary to inform regulatory policy formulation. In this study, we examine the microstructure of the surficial layers of sea ice exposed to oil using X-ray microtomography. Through analysis, 3D imaging of the spatial distribution of the ice's components (brine, air, and oil) were made. Additional quantitative information regarding the size, proximity, orientation, and geometry of oil inclusions were computed to ascertain discernable relationships between oil and the other components of the ice. Our results indicate implications for airborne remote sensing and bioremediation of the upper sea ice layers.

Photooxidation and biodegradation potential of a light crude oil in first-year sea ice.

Disappearing sea ice in the Arctic region results in a pressing need to develop oil spill mitigation techniques suitable for ice-covered waters. The uncertainty around the nature of an oil spill in the Arctic arises from the ice-covered waters and sub-zero temperatures, and how they may influence natural attenuation efficiency. The Sea-ice Environmental Research Facility was used to create a simulated Arctic marine setting. This paper focuses on the potential for biodegradation of the bulk crude oil content (encapsulated in the upper regions of the ice), to provide insight regarding the possible fate of crude oil in an Arctic marine setting. Cheaper and faster methods of chemical composition analysis were applied to the samples to assess for weathering and transformation effects. Results suggest that brine volume in ice may not be sufficient at low temperatures to encompass biodegradation and that seawater is more suitable for biodegradation.

Effect of dissolution, evaporation, and photooxidation on crude oil chemical composition, dielectric properties and its radar signature in the Arctic environment.

Accidental release of petroleum in the Arctic is of growing concern owing to increases in ship traffic and possible future oil exploration. A crude oil-in-sea ice mesocosm experiment was conducted to identify oil-partitioning trends in sea ice and determine the effect of weathering on crude oil permittivity. The dissolution of the lighter fractions increased with decreasing bulk oil-concentration because of greater oil-brine interface area. Movement of the oil towards the ice surface predominated over dissolution process when oil concentrations exceeded 1 mg/mL. Evaporation decreased oil permittivity due to losses of low molecular weight alkanes and increased asphaltene-resin interactions. Photooxidation increased the permittivity of the crude oil due to the transformation of branched aromatics to esters and ketones. Overall, the weathering processes influenced crude oil permittivity by up to 15%, which may produce sufficient quantifiable differences in the measured normalized radar cross-section of the ice.

Oil behavior in sea ice: Changes in chemical composition and resultant effect on sea ice dielectrics.

There has been increasing urgency to develop methods for detecting oil in sea ice owing to the effects of climate change in the Arctic. A multidisciplinary study of crude oil behavior in a sea ice environment was conducted at the University of Manitoba during the winter of 2016. In the experiment, medium-light crude oil was injected underneath young sea ice in a mesocosm. The physical and thermodynamic properties of the oil-infiltrated sea ice were monitored over a three-week time span, with concomitant analysis of the oil composition using analytical instrumentation. A resonant perturbation technique was used to measure the oil dielectric properties, and the contaminated sea ice dielectric properties were modeled using a mixture model approach. Results showed that the interactions between the oil and sea ice altered their physical and thermodynamic properties. These changes led to an overall decrease in sea ice dielectrics, potentially detectable by remote sensing systems.

Examining the physical processes of corn oil (medium crude oil surrogate) in sea ice and its resultant effect on complex permittivity and normalized radar cross-section.

Due to the effects of heightened warming in the Arctic, there has been an urgency to develop methods for detecting oil in (or under) sea ice, owing to increasing potential for oil exploration and ship traffic in the more accessible Arctic regions. To test the potential for radar utilizing the normalized radar cross section (NRCS) of the sea ice, an oil-in-ice mesocosm experiment was performed. Throughout the experiment, corn oil was used as a surrogate for medium crude oil, to assess oil movement tendencies in sea ice, and the resultant impact on the complex permittivity through measurement and modelling techniques. We performed a modelling study to establish the effects of corn oil on the NRCS of sea ice. The oil presence in the sea ice increased the temperature and reduced the salinity of the sea ice, thereby lowering its complex permittivity and modeled NRCS when compared to control sea ice.

Do estuaries pose a toxic contamination risk for wading birds?

The impact of potentially toxic chemicals on wildlife is commonly assessed by comparing the intake of the contaminant with the "no observable effects level" (NOAEL) of intake. It is known, however, that there are considerable uncertainties inherent in this method. This study presents a Monte-Carlo based model to assess the degree of risk posed to birds (dunlin, Calidris alpina) from important estuarine habitats, and to show the limitations of such risk assessments, particularly with regard to data availability. The model was applied to predict the uptake of metals (Hg, Pb) in this shorebird species in Poole Harbour and the Severn Estuary/Bristol Channel, UK, two internationally important shorebird habitats. The results show that in both areas, Pb and Hg concentrations may pose an ecologically relevant toxic risk to wading birds. For Pb, uncertainty in NOAEL values dominates the overall uncertainty. Use of lethal toxicity data (LD50/100) was investigated as a method for assessing sub-lethal impacts from Hg. It was found that this method led to a significant under-estimate of the potential impact of Hg contamination, compared with direct estimation of NOAEL.

When enough is not enough: shorebirds and shellfishing.

In a number of extensive coastal areas in northwest Europe, large numbers of long-lived migrant birds eat shellfish that are also commercially harvested. Competition between birds and people for this resource often leads to conflicts between commercial and conservation interests. One policy to prevent shellfishing from harming birds is to ensure that enough food remains after harvesting to meet most or all of their energy demands. Using simulations with behaviour-based models of five areas, we show here that even leaving enough shellfish to meet 100% of the birds' demands may fail to ensure that birds survive in good condition. Up to almost eight times this amount is needed to protect them from being harmed by the shellfishery, even when the birds can consume other kinds of non-harvested prey.

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.

A putative prokaryote voltage-gated Ca(2+) channel with only one 6TM motif per subunit.

Until now, voltage-gated Ca(2+) channel proteins have been found only in eukaryotes. Here we report that a gene recently discovered in the eubacterium Bacillus halodurans codes for a protein closely related to eukaryotic Ca(2+) channels, but that has only one 6-transmembrane-segement (6TM) motif, instead of four, in its pore-forming subunit. This is supported by the comparison of consensus sequences, which, along with the patterns of residue conservation, indicates a similar structure in the membrane to voltage-gated K(+) channels. From this we hypothesize that Ca(2+) channels originally evolved in bacteria, and that the specific eubacteria protein highlighted here is an ideal candidate for structure determination efforts.

Anisotropy of fluctuation dynamics of proteins with an elastic network model.

Fluctuations about the native conformation of proteins have proven to be suitably reproduced with a simple elastic network model, which has shown excellent agreement with a number of different properties for a wide variety of proteins. This scalar model simply investigates the magnitudes of motion of individual residues in the structure. To use the elastic model approach further for developing the details of protein mechanisms, it becomes essential to expand this model to include the added details of the directions of individual residue fluctuations. In this paper a new tool is presented for this purpose and applied to the retinol-binding protein, which indicates enhanced flexibility in the region of entry to the ligand binding site and for the portion of the protein binding to its carrier protein.

A family of putative Kir potassium channels in prokaryotes.

BACKGROUND: Prior to this report, members of the inward rectifier family, or Kir, have been found only in eukaryotes. Like most K+ channels, the pore-forming part of the protein is formed by four identical, or closely related, subunits. Each subunit contains a transmembrane M1-P-M2 motif that is followed by a relatively large C-terminus region unique to Kir's. RESULTS: In searching unfinished microbial genomes for K+ channels, we identified five sequences in the prokaryote Burkholderia pseudomallei, Burkholderia cepacia, Burkholderia fungorum LB400, Magentospirillum magnetotacticum, and Nostoc Punctiforme genomes that code for proteins whose closest relatives in current sequence databases are eukaryote Kir's. The sequence similarity includes the C-terminus portion of Kir's, for which there are no other close homologs in current prokaryote sequences. Sequences of the pore-forming P and M2 segments of these proteins, which we call KirBac, is intermediate between those of eukaryotic Kir's and several other K+ channel families. CONCLUSIONS: Although KirBac's are more closely related to Kir's than to other families of K+ channels, the intermediate nature of their pore-forming P and M2 segments suggests that they resemble an ancestral precursor to the eukaryotic Kir's. The similarity of KirBac to the bacterial KcsA channel, whose transmembrane structure has been solved, helps align Kir's with KcsA. KirBac's may assist in solving the three-dimensional structure of a member of the Kir family since bacterial membrane proteins are more easily expressed in the quantities necessary for crystallography.

Individual feeding specialisation in shorebirds: population consequences and conservation implications.

Individual feeding specialisation in shorebirds is reviewed, and the possilble mechanisms involved in such specialisations. Any specialisation can he seen as an individual strategy, and the optimum strategy for any given individual will be conditional upon its specific priorities and constraints. Some specialisations are related to social status and some to individual skills. Some are also probably frequency-dependent. However, most shorebird specialisations are constrained to a large extent by individual morphology, particularly bill morphology. For example, larger birds are able to handle larger prey, and birds with longer bills are able to feed on more deeply buried prey. Sex differences in bill length are uncommon in the Charardriidae, which are surface peckers, but are common in the Scolopacidae, which feed by probing in soft substrates. Sex differences in bill morphology are frequently associated with sex differences in feeding specialisation. There is evidence that different feeding specialisations are associated with different payoffs, in which case the probability of failing to reproduce or of dying will not be distributed equally throughout the population. I consider the population consequences of such feeding specialisations, particularly the different risks and benefits associated with different habitats or diets. I also consider the way in which individuals may differ in their response to habitat loss or change. I suggest that population models designed to predict the effect of habitat loss or change on shorebirds should have the ability to investigate the differential response of certain sections of the population, particularly different ages or sexes, that specialise in different diets or feeding methods.

Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels?

Evidence is presented that the transmembrane KdpA subunit of the high affinity K(+)-translocating P-type Kdp-ATPase is evolutionarily derived from the superfamily of 2TM-type K(+) channels in bacteria. This extends a previous study relating the K(+) channels to the KtrAB, Trk, Trk1,2, and HKT1 K(+) symporter superfamily of both prokaryotes and eukaryotes. Although the channels are formed by four single-MPM motif subunits, the transmembrane KdpA subunit and the transmembrane subunit of the symporter proteins are postulated to have four corresponding MPM motifs within a single sequence. Analysis of 17 KdpA sequences reveals a pattern of residue conservation similar to that of the symporters and channels, and consistent with the crystal structure of the KcsA K(+) channel. In addition, the most highly conserved residues between the families, specifically the central glycines of the P2 segments, are those previously identified as crucial for the property of K(+)-selectivity that is common to each protein. This hypothesis is consistent with an experimental study of mutations that alter K(+) binding affinity of the Kdp transporter. Although most of the results of a previous study of the transmembrane topology of KdpA are consistent with the 4-MPM model, the one deviation can be explained by a plausible change in the structure due to the experimental method.

Evolutionary relationship between K(+) channels and symporters.

The hypothesis is presented that at least four families of putative K(+) symporter proteins, Trk and KtrAB from prokaryotes, Trk1,2 from fungi, and HKT1 from wheat, evolved from bacterial K(+) channel proteins. Details of this hypothesis are organized around the recently determined crystal structure of a bacterial K(+) channel: i. e., KcsA from Streptomyces lividans. Each of the four identical subunits of this channel has two fully transmembrane helices (designated M1 and M2), plus an intervening hairpin segment that determines the ion selectivity (designated P). The symporter sequences appear to contain four sequential M1-P-M2 motifs (MPM), which are likely to have arisen from gene duplication and fusion of the single MPM motif of a bacterial K(+) channel subunit. The homology of MPM motifs is supported by a statistical comparison of the numerical profiles derived from multiple sequence alignments formed for each protein family. Furthermore, these quantitative results indicate that the KtrAB family of symporters has remained closest to the single-MPM ancestor protein. Strong sequence evidence is also found for homology between the cytoplasmic C-terminus of numerous bacterial K(+) channels and the cytoplasm-resident TrkA and KtrA subunits of the Trk and KtrAB symporters, which in turn are homologous to known dinucleotide-binding domains of other proteins. The case for homology between bacterial K(+) channels and the four families of K(+) symporters is further supported by the accompanying manuscript, in which the patterns of residue conservation are demonstrated to be similar to each other and consistent with the known 3D structure of the KcsA K(+) channel.

Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel.

Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function.

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting.

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36 degrees, whereas wt p53 bends it by 51-57 degrees. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by approximately 35 degrees and approximately 70 degrees, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

Structural models of the transmembrane region of voltage-gated and other K+ channels in open, closed, and inactivated conformations.

A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+ channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+ and Ca2+ channels. The role that our group plays in this process is to combine the information gained from experimental studies with molecular modeling techniques to generate atomic-scale structural models of these proteins. The modeling process involves three stages which are summarized as: (I) prediction of the channel sequence transmembrane topology, including the functionality and secondary structure of the segments; (II) prediction of the relative positions of the transmembrane segments, and (III) filling in all atoms of the amino acid residues, with conformations for energetically stabilized interactions. Both physiochemical and evolutionary principles (including sequence homology analysis) are used to guide the development. In addition to testing the steric and energetic feasibilities of different structural hypotheses, the models provide guidance for the design of new experiments. Structural modeling also serves to "fill in the gaps" of experimental data, such as predicting additional residue interactions and conformational changes responsible for functional processes. The modeling process is currently at the stage that experimental studies have definitely confirmed most of our earlier predictions about the transmembrane topology and functionality of different segments. Additionally, this report describes the detailed, three-dimensional models we have developed for the entire transmembrane region and important functional sites of the voltage-gated Shaker K+ channel in the open, closed, and inactivated conformations (including the ion-selective pore and voltage-sensor regions). As part of this effort, we also describe how our development of structural models for many of the other major K+ channel families aids in determining common structural motifs. As an example, we also present a detailed model of the smaller, bacterial K+ channel from Streptomyces lividans. Finally, we discuss strategies for using newly developed experimental methods for determining the structures and analyzing the functions of these channel proteins.

Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41.

We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus-1 (HIV-1) envelope glycoprotein-expressing cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol. We developed a new three-color assay to keep track of the cell into which fluorescent lipids and/or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipid-permissive fusion pore and a solute-permissive fusion pore (FPS), respectively. A synthetic peptide (DP178) corresponding to residues 643-678 of the HIV-1LAI gp120-gp41 sequence (Wild, C.T., D.C. Shugars, T.K. Greenwell, C.B. McDanal, and T.J. Matthews. 1994. Proc. Natl. Acad. Sci. USA. 91:12676-12680) completely inhibited FPS at 50 ng/ml, whereas at that concentration there was 20-30% fusion activity measured by the lipid redistribution. The differences detected in lipid mixing versus contents mixing are maintained up to 6 h of coculture of gp120-41-expressing cells with target cells, indicating that DP178 can "clamp" the fusion complex in the lipid mixing intermediate for very long time periods. A peptide from the NH2-terminal of gp41, DP107, inhibited HIV-1LAI gp120-gp41-mediated cell fusion at higher concentrations, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations. The inhibition of solute redistribution by DP178 was complete when the peptide was added to the fusion reaction mixture during the first 15 min of coculture. We have analyzed the inhibition data in terms of a fusion pore dilation model that incorporates the recently determined high resolution structure of the gp41 core.