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Kamarei F.,University of Tennessee at Knoxville | Gritti F.,University of Tennessee at Knoxville | Guiochon G.,University of Tennessee at Knoxville | Burchell J.,JASCO
Journal of Chromatography A | Year: 2014

The implementation of the traditional FA method is difficult with classical supercritical fluid chromatography (SFC) instruments. The instrument mixer and other sources of extra-column volumes are large and significantly broaden the fronts of injected plugs, which diminishes the precision and accuracy of the FA method. An SFC instrument was modified to permit more accurate determinations of adsorption isotherm data. The sample, the modifier, and CO2 are separately pumped via small volume connection tubes into a small volume mixer (250μL), where they are mixed into a homogeneous fluid fed to the column. The extra-column volumes and the column hold-up volume were accurately measured at each back pressure from the retention times of tracers. This modified instrument was used to measure the adsorption isotherm of S-naproxen by frontal analysis (FA) on a (R, R)-Whelk-O1 column, using a mixture of methanol (20%, v/v) and CO2 as the mobile phase. Its performance is studied at several different back pressures from 100 to 210bar. In all the experiments, the total flow rate was kept to a low value (1mL/min) in order to minimize the variation of the equilibrium constant along the column. Although a suitable breakthrough curve could not be obtained at low back pressures (<150bar) due to the closeness to the critical point pressure of the methanol/CO2 mixture, excellent results were obtained at higher back pressures (>150bar), conditions remote from the critical point and breakthrough curves with very sharp front shocks are obtained. The RSDs of the profiles recorded at each back pressures are excellent, better than 1%. © 2013 Elsevier B.V. Source

Boyd I.L.,University of St. Andrews | Frisk G.,Florida Atlantic University | Urban E.,University of Delaware | Tyack P.,Woods Hole Oceanographic Institution | And 22 more authors.
Oceanography | Year: 2011

The effect of noise on marine life is one of the big unknowns of current marine science. Considerable evidence exists that the human contribution to ocean noise has increased during the past few decades: human noise has become the dominant component of marine noise in some regions, and noise is directly correlated with the increasing industrialization of the ocean. Sound is an important factor in the lives of many marine organisms, and theory and increasing observations suggest that human noise could be approaching levels at which negative effects on marine life may be occurring. Certain species already show symptoms of the effects of sound. Although some of these effects are acute and rare, chronic sublethal effects may be more prevalent, but are difficult to measure. We need to identify the thresholds of such effects for different species and be in a position to predict how increasing anthropogenic sound will add to the effects. To achieve such predictive capabilities, the Scientific Committee on Oceanic Research (SCOR) and the Partnership for Observation of the Global Oceans (POGO) are developing an International Quiet Ocean Experiment (IQOE), with the objective of coordinating the international research community to both quantify the ocean sound scape and examine the functional relationship between sound and the viability of key marine organisms. SCOR and POGO will convene an open science meeting to gather community input on the important research, observations, and modeling activities that should be included in IQOE. © 2011 by The Oceanography Society. All rights reserved. Source

News Article
Site: http://www.nature.com/nature/current_issue/

The repeat module design process applied here consisted of an initial diversification round of large-scale sampling followed by filtering and clustering and then a second intensification round of sampling focused on successful topologies identified in the first round. Starting backbone models for sequence design were built using a fragment assembly protocol which is based on the standard Rosetta ab initio protocol31 with the following modifications: (1) fragment replacement moves were performed symmetrically across all repeats, guaranteeing that backbone torsion angles were identical at corresponding positions across repeats; (2) a pseudo-energy term (equal to the deviation between actual and desired curvature, in degrees, plus the deviation in rise multiplied by a factor of 5) was added to the potential to favour satisfaction of the geometric constraints; (3) the amino-acid sequence used for low-resolution scoring was assigned randomly at the start of each simulation from secondary-structure-specific distributions (helix: Ala+Ile+Leu+Asp+Ser; turn: Gly+Ser), which had the effect of increasing the diversity in helix packing distances and geometries compared with using a constant sequence such as poly-Val or poly-Leu. At the start of each independent design trajectory, the lengths of the secondary structure elements and turns were chosen randomly, defining the target secondary structure of the repeat module and its length. Together with the number of repeats, this defined the total length of the protein and the complete secondary structure, which was used to select 3- and 9-residue backbone fragments for use in the low-resolution fragment assembly phase. The design calculations reported here sampled helix lengths from 7 to 20 residues, turn lengths from 1 to 5 residues, and total repeat lengths ranging from 20 to 40 residues. The low-resolution fragment assembly simulation was followed by an all-atom sequence design stage consisting of two cycles alternating between fixed-backbone sequence design and fixed-sequence structure relaxation. Symmetry of backbone and side-chain torsion angles and sequence identities was maintained across all repeats. Since the starting backbones for design were built by relatively coarse sampling in a low-resolution potential, sequences designed with the standard all-atom potential were dominated by small amino acids and the resulting structures tended to be under-packed. To correct for this tendency, a softened Lennard–Jones potential32 was used for the sequence design steps, while the standard potential was used during the relaxation step. The Rosetta score12prime weights set was used as the standard potential for these design calculations. Final design models (typically 10,000–100,000 in this study) were first sorted by per-residue energy (total energy divided by the number of residues, to account for varying repeat length) and the top 20% filtered for packing quality (sasapack_score <0.5), satisfaction of buried polar groups (buried unsatisfied donors per repeat <1.5, buried unsatisfied acceptors per repeat <0.5), and sequence-structure compatibility via a fast, low-resolution symmetric refolding test (40 trajectories, requiring at least 1 under an r.m.s.d. threshold of 2 Å for 3-repeat designs and 4 Å for larger designs). Designs that passed these filters were clustered by C-α r.m.s.d. (allowing for register shifts when aligning helices with unequal lengths) to identify recurring architectures. The clusters were ranked by averaging residue energy, packing quality, and refolding success over all cluster members. During the intensification round of designs, representative topologies from successful design clusters were specifically resampled by enforcing their helix and turn lengths as well as their turn conformations (defined using a five-state, coarse-grained backbone torsion alphabet27; Extended Data Fig. 1e) during fragment selection. Selected low-energy designs from the second round that pass the filters described above were evaluated by a large-scale refolding test in which 2,000–10,000 ab initio models were built by standard (asymmetric) fragment assembly followed by all-atom relaxation. Success was measured by assessing the fraction of low-energy ab initio models with r.m.s.d. values to the design model under a length-dependent threshold. For designed toroids with an open, polar central pore, perfect symmetry may not allow optimal electrostatic interactions between nearby side chains corresponding to the same repeat position in successive repeats. We therefore explored symmetry-breaking mutations at a handful of inward-pointing positions via fixed-backbone sequence design simulations in which the length of the repeating sequence unit was doubled/tripled (for example, whereas perfect six-fold repeat symmetry would require K-K-K-K-K-K or E-E-E-E-E-E, doubling the repeat length allows charge complementarity with K-E-K-E-K-E). Solutions from these designs were accepted if they significantly lowered the total energy. The 12x31L design construct was generated by duplicating the final three repeats of the 9x31L design. To build a ‘design model’ for comparison with the experimentally determined structure, we followed the resampling protocol now forcing the 12x31L amino-acid sequence in addition to the number of repeats (12) and the helix and turn lengths (H14-L3-H11-L3) and turn conformations (GBB). Thus the sequence design steps were reduced to rotamer optimization (since the amino-acid identities were fixed). This symmetric structure prediction process was repeated 10,000 times and the lowest-energy final model was taken as the computational model. For a single representative of the 3x31L and 6x31R families, we performed lattice docking and design simulations to select mutations that might promote crystallization. Core positions were frozen at the design sequence. Candidate space groups were selected from those most commonly observed in the protein structural database. Theoretical models of crystal packing arrangements were built by randomly orienting the design model within the unit cell and reducing the lattice dimensions until clashes were encountered. Symmetric interface design was performed on these docked arrangements, and final designs were filtered by energy, packing, satisfaction of polar groups, and number of mutations from the original design model. To compute the handedness of helical bundles formed by tandem repeat proteins, we generated an approximate helical bundle axis curve by joining the location of repeat-unit centres of mass in a sliding fashion along the protein chain. The handedness was then estimated by computing the directionality of the winding of the polypeptide chain about this axis curve. To assess similarity between design models and proteins in the structural database, we performed searches using the structure–structure comparison program DALI33 as well as consulting the protein structure classification databases CATH34, SCOPe35, and ECOD36. Further details are given in Supplementary Discussion. Repeat protein design methods were implemented in the Rosetta software suite (www.rosettacommons.org) and will be made freely available to academic users; licenses for commercial use are available through the University of Washington Technology Transfer office. The plasmids encoding individual constructs were cloned into previously described bacterial pET15HE expression vectors37 containing a cleavable N-terminal His-tag and an ampicillin resistance cassette. Sequence-verified plasmids were transformed into BL21(DE3)RIL Escherichia coli cells (Agilent Technologies) and plated on lysogeny broth (LB) medium with ampicillin (100 μg ml−1). Colonies were individually picked and transferred to individual 10 ml aliquots of LB–ampicillin media and shaken overnight at 37 °C. Individual 10 ml aliquots of overnight cell cultures were added to individual 1 l volumes of LB–ampicillin, which were then shaken at 37 °C until the cells reached an absorbance at 600 nm of 0.6–0.8. The cells were chilled for 20 min at 4 °C, then isopropyl-β-D-thiogalactoside (IPTG) was then added to each flask to a final concentration of 0.5 mM to induce protein expression. The flasks were shaken overnight at 16 °C, and then pelleted by centrifugation and stored at − 20 °C until purification. Construct dTor_6x35L(SeMet), incorporating a single methionine residue at position 168 in the original design construct, was generated using a QuikChange site-directed mutagenesis kit (Agilent) and corresponding protocol from the vendor. The resulting plasmid construct was again transformed into BL21(DE3)RIL E. coli cells (Agilent Technologies) and plated on LB plates containing ampicillin (100 μg ml−1) and chloramphenicol (35 g ml−1). Subsequent cell culture and protein expression in minimal media, along with incorporation of selenomethionine, was incorporated during protein expression according to ref. 38. Cell pellets from 3 l of cell culture were resuspended in 60 ml of PBS solution (140 mM NaCl, 2.5 mM KCl, 10 mM NaHPO , 2 mM KH PO ) containing 10 mM imidazole (pH 8.0). Cells were lysed via sonication and centrifuged to remove cell debris. The supernatant was passed through a 0.2 μm filter, and then incubated on a rocker platform at 4 °C for 1 h after adding 3 ml of resuspended nickel-NTA metal affinity resin (Invitrogen). After loading onto a gravity-fed column, the resin was washed with 45 ml of the same lysis buffer described above, and the protein was eluted from the column with three consecutive aliquots of PBS containing 150 mM imidazole (pH 8.0). Purified protein was concentrated to approximately 5–25 mg ml−1 while buffer exchanging into 25 mM Tris (pH 7.5) and 200 mM NaCl and then further purified via size-exclusion chromatography using HiLoad 16/60 Superdex 200 column (GE). Protein samples were then split in half; one sample was used directly for crystallization while the other had the His tag removed by an overnight digest with biotinylated thrombin (Novagen), before additional crystallization trials. The digested sample was incubated for 30 min with streptavidin-conjugated agarose (Novagen) to remove the thrombin. All samples were tested for purity and removal of the His tag via SDS–polyacrylamide gel electrophoresis. The final protein samples, both with and without the N-terminal poly-histidine affinity tag, were concentrated to values of 5–25 mg ml−1 for crystallization trials. Proteins at a concentration of 4–10 mg ml−1 were run over a Superdex 75 10/300 GL column (GE Healthcare) in 25 mM Tris pH 8.0 plus 100 or 750 mM NaCl at a rate of 0.4 ml min−1 on an AKTAprime plus chromatography system (GE Healthcare). All fractions containing eluted toroid protein (visualized via electrophoretic gel analyses) were pooled, concentrated, and run over the column a second time to assess their solution oligomeric behaviour using protein with a minimal background of contaminants. Gel filtration standards (Bio-Rad) were run over the same column in matching buffer, and the ultraviolet trace of the proteins was overlaid onto the standards using UNICORN 5 software (GE Healthcare). For measurements of protein stability using circular dichroism spectroscopy, purified recombinant toroid constructs were diluted to between 10 and 20 μM concentration and dialysed overnight into 10 mM potassium phosphate buffer at pH 8.0. Circular dichroism thermal denaturation experiments were performed on a JASCO J-815 circular dichroism spectrometer with a Peltier thermostat. Wavelength scans (190–250 nm) were performed for each construct at 20 °C and 95 °C. Additional thermal denaturation experiments were conducted by monitoring circular dichroism signal strength at 206 nm over a temperature range of 4–95 °C (0.1 cm path-length cell), with measurements taken every 2°. Sample temperature was allowed to equilibrate for 30 s before each measurement. Purified proteins were initially tested for crystallization via sparse matrix screens in 96-well sitting drops using a mosquito (TTP LabTech). Crystallization conditions were then optimized with constructs that proved capable of crystallizing in larger 24-well hanging drops. Out of 11 constructs that were purified to homogeneity, 10 were crystallized, of which 5 yielded high quality X-ray diffraction that resulted in successful structure determination. dTor_6x35L was crystallized in 160 mM sodium chloride, 100 mM Bis-Tris pH 8.5 and 24% (w/v) polyethylene glycol 3350 at a concentration of 26 mg ml−1. The crystal was transferred to a solution containing 300 mM, then 500 mM sodium chloride and flash frozen in liquid nitrogen. Data were collected on a R-AXIS IV++ at wavelength 1.54 Å and processed on an HKL2000 (ref. 39). dTor_6x35L(SeMet) was crystallized in 140 mM sodium chloride, 100 mM Tris pH 8.5 and 22% (w/v) polyethylene glycol 3350 at a concentration of 26 mg ml−1. The crystal was transferred to a solution containing 300 mM, then 500 mM sodium chloride and flash frozen in liquid nitrogen. Data were collected at ALS Beamline 5.0.2 at wavelength 0.9794 Å and processed on an HKL2000 (ref. 39). dTor_3x33L_2-2 was crystallized in two different conditions, producing two different crystal lattices. The first condition had 30% polyethylene glycol 3350, 100 mM Tris pH 6.5, 200 mM NaCl with a protein concentration of 1.8 mM. The protein was soaked in a 15% ethylene glycol cryoprotectant for 1 min before being flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ (Rigaku) at wavelength 1.54 Å for 180° at φ = 0 and another 180° at φ = 180. Data were then processed on an HKL2000 (ref. 39) out to 1.85 Å in space group P2 2 2 . The second condition had 45% polyethylene glycol 400 and 100 mM Tris pH 7.7 with a protein concentration of 1.8 mM. Protein crystal was flash frozen without being cryoprotected. Data were collected on a Saturn 944+ (Rigaku) at wavelength 1.54 Å for 180° at φ = 0 and another 180° at phi = 180. Data were then processed on an HKL2000 (ref. 39) out to 1.85Å in space group P4 2 2. dTor_9x31L_sub was crystallized in 100 mM Tris pH 8.5 and 15% (v/v) ethanol at a concentration of 11.5 mg ml−1. The crystal was transferred to a solution containing 75 mM Tris pH 8.5, 7.5% (v/v) ethanol and 25% (v/v) glycerol and flash frozen in liquid nitrogen. Data were collected at ALS Beamline 5.0.2 at wavelength 1.0 Å and processed on an HKL2000 (ref. 39) out to 2.9 Å in space group P4 2 2/P4 2 2. dTor_9x31L was crystallized in 0.1 M sodium citrate pH 5.4 and 1.0 M ammonium phosphate monobasic at a concentration of 8.8 mg ml−1 in 3 μl drops containing 1 μl protein and 2 μl well solution. The crystal was transferred to a solution containing the well plus 25% (v/v) glycerol and flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ charge-coupled device at wavelength 1.54 Å and processed on an HKL2000 (ref. 39) out to 2.5 Å in space group P2 2 2 . dTor_12x31L was crystallized in 0.9 M sodium malonate pH 7.0, 0.1 M HEPES pH 7.0 and 0.5% Jeffamine ED-2001 pH 7.0 at a concentration of 8.8 mg ml−1 in 2 μl drops containing 1 μl protein and 1 μl well solution. The crystal was transferred to a solution containing 0.675 M sodium malonate pH 7.0, 0.075 M HEPES pH 7.0, 0.375% Jeffamine ED-2001 pH 7.0 and 25% glycerol, and flash frozen in liquid nitrogen. Data were collected on a Saturn 944+ charge-coupled device at wavelength 1.54 Å and processed on an HKL2000 (ref. 39) out to 2.3 Å in space group R3:H. The dTor_6x35L and both dTor_3x33L_2-2 structures were solved by Molecular Replacement with Phaser40 via CCP4i41 using the Rosetta-designed structure as a search model. The structures were then built and refined using Coot42 and Refmac543, respectively. The structure of dTor_6x35L(SeMet) was solved by Molecular Replacement with Phaser40 via PHENIX44 using the best refined model of dTor_6x35L as a phasing model. The structure was then built and refined using Coot42 and PHENIX45, respectively. The structures of dTor_9x31L_sub and dTor_9x31L were solved by Molecular Replacement with Phaser40 via PHENIX44 using the Rosetta-designed structure as a search model. The structure was then built and refined using Coot42 and PHENIX45, respectively. The structure of dTor_12x31L was solved by Molecular Replacement with Phaser40 via PHENIX44 using a 4-repeat subunit the Rosetta-designed structure as a search model. The structure was then built and refined using Coot42 and PHENIX45, respectively. Final Ramachandran statistics after refinement were as follows (given as % preferred, % allowed, % outliers, respectively): dTor_6x35L(SeMet): 98.06, 1.94, 0.0; dTor_3x33L_2-2a: 99.48, 0.0, 0.52; dTor_3x33L_2-2b: 98.96, 0.52, 0.52; dTor_9x31L_sub: 98.31, 1.69, 0.0; dTor_9x31L: 99.28, 0.36, 0.36; dTor_12x31L: 99.0, 1.0, 0.0.

Ishikawa M.,Osaka University | Katsura M.,Osaka University | Nakashima S.,Osaka University | Aizawa K.,JASCO | And 3 more authors.
e-Journal of Surface Science and Nanotechnology | Year: 2011

The goal of the present study is to obtain broadband near-field infrared (IR) spectra by combining Fouriertransform infrared spectroscopy (FTIR) with scattering near-field optical microscopy (s-SNOM). A stage was added to the IR spectrometer with a ceramic light source in order to modulate the probe-sample distance, and the second harmonic component was extracted by a lock-in amplifier. The detected IR signal intensity decreased exponentially with the distance between the probe tip and an Au mirror, with a localization scale of approximately 100 nm. An area with Au islands formed by electron beam lithography was scanned with the modulation system with mapping steps of X = 80 nm and Y = 133 nm. The obtained IR intensity image matches the topographic image, indicating sub-micron spatial resolution. These results indicate that the addition of the modulation system to the broadband near-field IR spectrometer was successful in obtaining localized near-field signals and sub-micron spatial resolution, even using a ceramic IR light source. © 2011 The Surface Science Society of Japan. Source

Mangabhai R.,Mangabhai Consulting | Cave S.,JASCO | Wills R.,JASCO
Concrete (London) | Year: 2010

As part of CE marking under EN 1504 Parts 2 and 3(1), identification tests using BS EN 1767:1999(2) are required on the products at yearly intervals. Source

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