An investigation of a polymetamorphic terrain using ⁴⁰Ar-³⁹Ar geochronology

This research is an exploration both of the Precambrian metamorphic geology of south-western Montana and the Wlaser ablation microprobe 40At-39At geochronological technique. Using the high spatial resolution of the UV laser microprobe it has been possible to produce systematic 40At-39Ar ages within individual mica grains. Further, the UV laser has been used to drill into biotite inclusions within garnet to look at the shielding effect of the garnet lattice, and to drill depth profiles into garnet to measure helium diffusion in laboratory experiments. The south-western Montana region of the Precambrian Wyoming Province was believed to have undergone at least three separate periods of metamorphism: M1- granulite facies in the Archaean, M2 - amphibolite facies in the Early Proterozoic, and M3 - greenschist to epidote-amphibolite facies in the Mid-Proterozoic. Because of the relatively low blocking temperature of the 40Ar- 39Ar geochronological system in mica (c.300-350 °C), it is readily reset by regional metamorphic events and previous K-Ar and 4OAr_38 Atgeochronology had found that the Early Proterozoic event dominated the ages obtained. UV laser 4OAt-39At dating of the matrix mica constrained the timing of cooling from the Early Proterozoic metamorphic event to between 1780 to 1740 Ma with a cooling rate between 1 and 8 °C/m.y. 40Ar-39Ar analyses of individual biotite inclusions in garnet also produced similar ages. However, Pb-Pb step leach dating of a small subset of garnet yielded ages between 1808 and 1765 Ma, demonstrating that the garnet did not grow during an Archaean event but, rather, during the Early Proterozoic metamorphic event. Thus, the shielding properties of garnet on biotite inclusions could not be easily assessed in these samples. However, where matrix biotite had been partially reset by the Cretaceous plutons, there was some evidence to suggest that a minority of the biotite inclusions in the same sample had been shielded from resetting. The influence of fractures, defects and other fast diffusion paths is believed to have prevented most of the inclusions from being shielded. No evidence was found to show that the rocks in south-western Montana were metamorphosed during the Archaean and it seems likely that the M1 and M2 events were not greatly separated in time and were both Early ProterozoiC in age. The high spatial resolution of the UV laser microprobe was used in order to date highly altered biotite within rocks that had undergone later (M3) greenschist metamorphism. Biotite was interlayered with chlorite, clinozoisite and prehnite but using the UV laser it was possible to separately analyse areas of unaltered biotite and areas of alteration within a single mineral and produce ages from both. It was thus possible to measure two ages from one sample: an unaltered age consistent with the timing of the Early Proterozoic metamorphism, and a younger age that could be linked to the c.1500-1360 Ma timing of Belt basin formation to the north and west of the region. This technique therefore was able to overcome the difficulties associated with producing meaningful ages from altered samples whilst constraining the timing of the M3 metamorphic event

3D structure of a Brucella melitensis porin: molecular modelling in lipid membranes

Brucella melitensis is a pathogenic bacterium responsible for brucellosis in mammals and humans. Its outer membrane proteins (Omp) control the diffusion of solutes through the membrane, and they consequently have a crucial role in the design of diagnostics and vaccines. Moreover, such proteins have recently revealed their potential for protein-based biomaterials. In the present contribution, the structure of the B. melitensis porin Omp2a is built using the RaptorX threading method. This is a 16-stranded ß-barrel with an a-helix on the third loop folding inside the barrel and forming the constriction zone of the channel, a typical feature of general porins such as PhoE and OmpF. The preferential diffusion of cations over anions experimentally observed in anterior studies is evidenced by the presence of distinct clusters of charges in the extracellular loops and in the inner pore. Docking studies support the previously reported hypothesis of Omp2a ability to aid maltotetraose diffusion. The monomer model is then assembled into a homotrimer, stabilized by the L2 loop involved in most of the interface interactions. The stability of the trimer is evaluated in three bilayers: pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and a mixture of 1:1 of POPC/POPE. All-atom molecular dynamics simulations demonstrate the ß-barrel-structural stability over time even though a breathing-like motion is observed. Compared to the pure bilayers, the POPC/POPE better preserves the integrity of the protein and its channel. Overall, this work demonstrates the relevancy of the Omp2a model and will help to design new therapeutic agents and bioinspired nanomaterialsPeer ReviewedPostprint (author's final draft

Structural and functional characterization of Solanum tuberosum VDAC36

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum , stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.Peer ReviewedPostprint (author's final draft

Thermomechanical Response of a Representative Porin for Biomimetics

The thermomechanical response of Omp2a, a representative porin used for the fabrication of smart biomimetic nanomembranes, has been characterized using microcantilever technology and compared with standard proteins. For this purpose, thermally induced transitions involving the conversion of stable trimers to bigger aggregates, local reorganizations based on the strengthening or weakening of intermolecular interactions, and protein denaturation have been detected by the microcantilever resonance frequency and deflection as a function of the temperature. Measurements have been carried out on arrays of 8-microcantilevers functionalized with proteins (Omp2a, lysozyme and bovine serum albumin). To interpret the measured nanofeatures, the response of proteins to temperature has been also examined using other characterization techniques, including real time wide angle X-ray diffraction. Results not only demonstrate the complex behavior of porins, which exhibit multiple local thermal transitions before undergoing denaturation at temperatures higher than 105 °C, but also suggest a posttreatment to control the orientation of immobilized Omp2a molecules in functionalized biomimetic nanomembranes and, thus, increase their efficacy in ion transport.Peer Reviewe

Properties of Omp2a-based supported lipid bilayers: comparison with polymeric bioinspired membranes

Omp2a ß-barrel outer membrane protein has been reconstituted into supported lipid bilayers (SLBs) to compare the nanomechanical properties (elastic modulus, adhesion forces, and deformation) and functionality of the resulting bioinspired system with those of Omp2a-based polymeric nanomembranes (NMs). Protein reconstitution into lipid bilayers has been performed using different strategies, the most successful one consisting of a detergent-mediated process into preformed liposomes. The elastic modulus obtained for the lipid bilayer and Omp2a are ~19 and 10.5 ± 1.7 MPa, respectively. Accordingly, the protein is softer than the lipid bilayer, whereas the latter exhibits less mechanical strength than polymeric NMs. Besides, the function of Omp2a in the SLB is similar to that observed for Omp2a-based polymeric NMs. Results open the door to hybrid bioinspired substrates based on the integration of Omp2a-proteoliposomes and nanoperforated polymeric freestanding NMs.Peer ReviewedPostprint (author's final draft

Purification and Characterization of Trehalase From Acyrthosiphon pisum, a Target for Pest Control.

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs (179)PGGRFRELYYWDTY(192) and (479)QWDFPNAWPP(489), a glycine-rich region (549)GGGGEY(554), and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with K(m) and V(max) values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health

Self-standing, conducting and capacitive biomimetic hybrid nanomembranes for selective molecular ion separation

Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kO cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.L-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kO cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.L-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.Peer ReviewedPostprint (author's final draft