Spiral Assembly Research Articles

Analysis of the Costructure of the Simian Virus 40 T-Antigen Origin Binding Domain with Site I Reveals a Correlation between GAGGC Spacing and Spiral Assembly

Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag). The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed.

Oligo(p‐phenylene‐ethynylene)‐Derived Super‐π‐Gelators with Tunable Emission and Self‐Assembled Polymorphic Structures

Linear π-conjugated oligomers are known to form organogels through noncovalent interactions. Herein, we report the effect of π-repeat units on the gelation and morphological properties of three different oligo(p-phenylene-ethynylene)s: OPE3, OPE5, and OPE7. All of these molecules form fluorescent gels in nonpolar solvents at low critical gel concentrations, thereby resulting in a blue gel for OPE3, a green gel for OPE5, and a greenish yellow gel for OPE7. The molecule-molecule and molecule-substrate interactions in these OPEs are strongly influenced by the conjugation length of the molecules. Silicon wafer suppresses substrate-molecule interactions whereas a mica surface facilitates such interactions. At lower concentrations, OPE3 formed vesicular assemblies and OPE5 gave entangled fibers, whereas OPE7 resulted in spiral assemblies on a mica surface. At higher concentrations, OPE3 and OPE5 resulted in super-bundles of fibers and flowerlike short-fiber agglomerates when different conditions were applied. The number of polymorphic structures increases on increasing the conjugation length, as seen in the case of OPE7 with n=5, which resulted in a variety of exotic structures, the formation of which could be controlled by varying the substrate, concentration, and humidity.

Cellulose Nanocrystal Reinforced Alginate Fibers—Biomimicry Meets Polymer Processing

This research takes a biomimetic approach to the design of polymer nanocomposites and demonstrates structure-property relationships that are controllable via processing conditions. Cellulose nanocrystals (CNCs) measuring 130 nm (length) × 20.4 nm (width) × 6.8 nm (height) were isolated from cotton by sulfuric acid hydrolysis and were incorporated in an alginate fiber wet spinning dope solution. Incorporating CNCs within the alginate fiber enables a nearly two-fold increase in the apparent jet stretch (JA ), ratio of the linear draw speed to extrusion velocity. Fiber spinning at a constant JA resulted in an unexpected decrease in fiber modulus and increase in toughness. Alternatively, fiber spinning at the maximum JA resulted in modulus increases that are predicted by the Halpin-Tsai model and the Hui-Shia model. Wide-angle X-ray diffraction (WAXD) was used to elucidate the structure and orientation of cellulose nanocrystals (CNC) within the alginate nanocomposite fibers and provide correlations with mechanical property enhancements. The spread of the azimuthal intensity distribution of the CNC (2,0,0) reflection increased with higher CNC loads until the nanoparticles within the matrix spiraled around the longitudinal axis. The appearance of a spiral angle with increasing CNC load resulted in a step reduction in modulus and increase in toughness. Increased fiber stretching during spinning retarded the appearance of the spiral assembly and increased CNC alignment. This spiral orientation is also observed in native cellulose fibers as a microfibril angle and is deterministic of their mechanical properties.

An FT-IR Study on Packing Defects in Mixed β-Aggregates of Poly(l-glutamic acid) and Poly(d-glutamic acid): A High-Pressure Rescue from a Kinetic Trap

Under favorable conditions of pH and temperature, poly(L-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of β(2)-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β(1)), the amide I' vibrational band of β(2)-fibrils is unusually red-shifted below 1600 cm(-1), which has been attributed to bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I' band of aggregates precipitating from racemic mixtures of PLGA and PDGA (β(1)) is dominated by components at 1613 and 1685 cm(-1)-typically associated with intermolecular antiparallel β-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The β(1)-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β(2)-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils.

Spiral Assembly of the 1D Chain Sheet of Fe(NCBH3)2(bpa)2·(biphenyl) (bpa = 1,2-bis(4-pyridyl)ethane) and its Stepwise Spin-Crossover Phenomenon

The assembled complex, Fe(NCBH3)2(bpa)2, enclathrating biphenyl has been synthesized, the skeleton of which is a 1D chain. Several 1D chains gather together to form 1D chain sheet. The 1D chain sheet is stacked spirally to form novel spiral assembly. The average Fe-N distances were 2.020(2), 2.083(2), and 2.191(2) Å at 100, 175, and 298 K, respectively, reflecting spin-state change of the iron ion. The assembly showed a stepwise spin-crossover phenomenon.

Organic high ionic strength aqueous two-phase solvent system series for separation of ultra-polar compounds by spiral high-speed counter-current chromatography.

Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar compounds, but often fail to provide suitable partition coefficient values for highly polar compounds, such as sulfonic acids, catecholamines and zwitter ions. The present paper introduces a new solvent series which can be applied for the separation of these polar compounds. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios and consists of a series of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s) can be found in a few steps. Each solvent system gives proper volume ratio and high density difference between the two phases to provide a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartrazine (sulfonic acid), tyrosine (zwitter ion) and epinephrine (a catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. The capability of the method is demonstrated by separation of three catecholamines.

Wide-Angle X-ray Diffraction of Cellulose Nanocrystal−Alginate Nanocomposite Fibers

Wide-angle X-ray diffraction (WAXD) was used to determine the structure and orientation of cellulose nanocrystals (CNC) within alginate nanocomposite fibers and provide correlations with the resulting mechanical property enhancements [Urena-BenavidesLangmuir 2010, 26, 14263]. The alginate junction zones were mostly composed of mannuronate/guluronate (MG) dimers; no evidence was found of an alteration of this structure upon addition of the nanofiller. The orientation of the CNC was studied from the azimuthal intensity distribution of the (2,0,0) reflection. The spread of the orientation increased with higher loads until the nanoparticles within the matrix spiraled around the longitudinal axis, which resulted in a reduction in tenacity and modulus and an increase in elongation at break and toughness. This spiral orientation is also observed in native cellulose fibers and is deterministic of their mechanical properties. Increased fiber stretching during spinning retarded the appearance of a spiral assembly a...

Spiral Countercurrent Chromatography Studies Using the Spiral Disk Assembly

The first rotor proposed for high speed countercurrent chromatography (HSCCC), the spiral disk assembly, consisting of a stack of 8 high density polyethylene plates with spiral flow channels, has been studied with various solvents and peptide separations to understand its application to polar compounds and solvent systems. Mostly polar solvent systems comprised of the heavy alcohols, were studied because of their suitability for peptide and larger molecule separation. Peptide mixtures were separated to correlate partition coefficients ranging from 0.3 to 2.8 with elution in spiral CCC. With some peptides, scale-up purification experiments were performed with sample loadings up to 85 mg in the 153 mL volume rotor. These studies characterized polar solvent systems with high stationary phase retentions that can be used for separation and purification of compounds with suitable partition coefficients. The solvent system, sec-butanol-1% aq.trifluoroacetic acid with a 73.3% stationary phase retention proved to be useful for most separations. This is a very useful method to substitute for the use of acetonitrile in preparative chromatography.

Spiral column configuration for protein separation by high-speed countercurrent chromatography

Retention of the stationary phase of aqueous–aqueous polymer phase systems is improved by a spiral column configuration which utilizes the radially acting centrifugal force along the spiral pitch to retain the heavier phase in the outer portion and the lighter phase in the inner portion of the spiral channel. For the separation of proteins which has low mass transfer rates, the system needs further modification of the separation channel to interrupt the laminar flow and enhance mixing of the two phases. Two spiral column assemblies were developed, one using a disk with spiral grooves and the other, the spiral tube support which accommodates the multiple spiral layers made from a single piece of fluorinated plastic tubing. In the spiral disk assembly, the best protein separation is achieved by the mixer–settler system which vigorously mixes two phases by vibrating glass beads placed in every other section of barricaded spiral channel, while in the spiral tube assembly the partition efficiency of proteins is enhanced by compressing the tubing to interrupt the laminar flow of the mobile phase. In both systems protein samples were well resolved by choosing the suitable elution modes.

Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic–aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems, such as chloroform–methanol–water (4:3:2) and hexane–ethyl acetate–methanol–water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, S f > 70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol–acetic acid–water (4:1:5) (<3 mL/min, S f > 70%), 12.5% poly(ethylene glycol) (PEG) 1000–12.5% K 2HPO 4–75% water (≤1 mL/min, S f > 70%) and 4% PEG 8000–5% Dextran T500–91% water (≤0.5 mL/min, S f > 50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.

Mixer-settler counter-current chromatography with multiple spiral disk assembly

A novel system for performing high-speed counter-current chromatography has been developed for separation of biopolymers using polymer phase systems. The spiral disk assembly consisting of eight units, each equipped with over 300 mixer-settler sets, was constructed and performance evaluated in terms of retention of the stationary phase and separation efficiency. A series of experiments was performed with a polymer phase system composed of polyethylene glycol 1000 (12.5%, w/w) and dibasic potassium phosphate (12.5%, w/w) using two stable protein samples of myoglobin and lysozyme at various experimental conditions of flow rates and revolution speeds. The best results were obtained with revolution speeds of 800–1000 rpm at flow rates of 0.25–0.5 ml/min where the partition efficiency of several 100 theoretical plates was achieved with over 50% stationary phase retention.

Mixer-settler counter-current chromatography with a barricaded spiral disk assembly with glass beads

A novel spiral disk is designed by placing barricades at 6 mm intervals in the middle of the spiral channel to divide the channel into multiple sections. Glass beads are placed in every other section so that the planetary motion produces repetitive mixing and settling of polymer phase systems. Performance of this mixer-settler spiral disk assembly was examined for separation of lysozyme and myoglobin with a polymer phase system. The best results were obtained with a spiral disk equipped with barricades with openings ranging from 1.2 to 0.4 mm on each side at a high revolution speed up to 1200 rpm.

Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the beta-sheets that span the intermolecular interfaces, with smaller but similar contributions from beta-sheets that span the intramolecular interfaces within each subunit. With the integrity of these beta-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.

Crystal Structure of the Membrane Fusion Protein, MexA, of the Multidrug Transporter in Pseudomonas aeruginosa

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 A. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod alpha-helical domain was globular and constructed by a cluster of eight short beta-sheets. The third domain located distal to the alpha-helical rod was globular and composed of seven short beta-sheets and one short alpha-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic alpha-barrel domain of OprM via the alpha-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

The Cyanide Degrading Nitrilase from Pseudomonas stutzeri AK61 Is a Two-Fold Symmetric, 14-Subunit Spiral

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.

Improved spiral disk assembly for high-speed counter-current chromatography

The spiral disk for type-J high-speed counter-current chromatography (HSCCC) has been improved by placing short segments of PTFE (tetrafluoroethylene) tubing into the channel at regular intervals. The best results were obtained with a four-channel spiral column using 600 spacers, which significantly improved stationary phase retention and partition efficiency in both n-butanol–acetic acid–water (4:1:5) and poly(ethylene glycol) 1000-dibasic phosphate polymer phase systems. Based on these findings, a “bead-chain spiral disk” was designed and its performance tested with a type-J HSCCC centrifuge. This new column design substantially improved the separation of both dipeptide and protein samples in terms of stationary phase retention and partition efficiency.

Spiral Disk Assembly for HSCCC: Column Design and Basic Studies on Chromatographic Resolution and Stationary Phase Retention

A set of four separation disks equipped with spiral channel(s) was designed for our type‐J high speed countercurrent chromatography (J‐HSCCC) centrifuge to improve retention of the stationary phase of polar solvent systems. Four different spiral disks were tested: two had a single spiral channel with different depths and the other two had four spiral channels connected in series to provide a greater spiral pitch. Performance of each disk was tested in terms of chromatographic resolution and/or stationary phase retention using three different two‐phase solvent systems, including 1‐butanol/acetic acid/water (4:1:5, v/v/v) for dipeptide separation; 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate for protein separation; and 4% (w/w) PEG8000–5% (w/w) dextran T500 in 10 mM dibasic sodium phosphate for determination of stationary phase retention. The results show that the spiral column retains a satisfactory amount of stationary phase for all solvent systems, even at a relatively high mobile phase flow rate where the spiral pitch plays a significant role. Separation of dipeptides with the butanol solvent system was possible using 10 mL/min flow rate of the mobile phase and 4‐spiral‐disks with their greater spiral pitch. In protein separations with the PEG–phosphate system, the single‐spiral disks yielded the best separation using the upper phase in tail‐to‐head elution mode at 1 mL/min, while the 4‐spiral disks show higher retention of the stationary phase. The retention of the PEG–dextran system is improved in the 4‐spiral disks, which exceed 60% at a flow rate of 0.5 mL/min. Various parameters, which affect the performance of the spiral disk separation column are discussed.

PROTEIN SEPARATION BY CROSS-AXIS COIL PLANET CENTRIFUGE WITH SPIRAL COLUMN ASSEMBLIES

ABSTRACT A newly fabricated spiral column assembly was first applied to the countercurrent chromatographic separation of proteins using the cross-axis coil planet centrifuge (cross-axis CPC). The separation was performed by a set of stable proteins such as cytochrome C, myoglobin, and lysozyme with an aqueous–aqueous polymer phase solvent system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate. Three sets of left-handed single-layer spiral columns with different IDs (1.0, 1.5, and 2.0 mm) were employed to investigate the effect of column ID on partition efficiency at four different elution modes. The elution modes consisted of the combinations of the direction of revolution (PI=counterclock-wise; PII=clockwise), the head–tail elution mode (H=head to tail; T=tail to head), and the inward–outward elution mode (I=inward; O=outward). Among these experiments, the best separation of proteins was attained using a 1.5 mm-ID column assembly with the PI-H-O elution mode of the lower phase mobile. The resolution between cytochrome C and myoglobin peaks was 0.9 and between myoglobin and lysozyme peaks was 1.0, while the retention of the stationary phase was 32.7%. In order to improve the partition efficiency, the four-layer spiral column assembly with 1.0 mm-ID column tubing was applied to the protein separation. The resolution between these peaks was improved to 1.1 and 1.2, respectively, while the stationary phase retention decreased to 20.9%. Further studies using a single-layer spiral coiled column also revealed the effective peak resolution of proteins, but with lower stationary phase retention. The overall results demonstrated the spiral column assembly was useful for the protein separation using the cross-axis CPC with the aqueous-aqueous polymer phase system.

Self-organized pattern formation in the oxidation of supported iron thin films. II. A simulation study

The process of oxidation of supported iron thin films is modeled by casting it into the form of an activator-inhibitor system, with precursor oxidation state as the activator, and stress produced by the large density difference between the metal and its oxide as a fast-diffusing inhibitor. An activator-substrate mechanism also coexists due to the finite availability of iron. The redistribution of iron by diffusion via vacancies also indirectly contributes to the activation process. A slow process of ripening, which minimizes surface energy, is suggested to convert the early leaflike pattern to a spiral assembly of hillocks. This model simulation yields patterns, which closely resemble the patterns observed in experiments reported by Shinde et al. [Phys. Rev. B 64, 035408 (2001)], in the accompanying Paper I.

Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals

Vinblastine, vincristine, vindesine, and vinorelbine, the four vinca alkaloids used in cancer therapy, differ in their antitumoral spectra and toxicities, but not in their inhibitory effects on microtubule assembly in vitro. At higher drug concentrations, vinca alkaloids induce the assembly of spiral filaments of tubulin, which, in turn, can interact laterally and form paracrystals. Using methods that distinguish spiral filaments and paracrystals (aggregated spirals), we found that spiral filament formation was largely independent of the incubation temperature, of the alkaloid used, and of the presence or absence of microtubule-associated proteins (MAPs). In contrast, the formation of aggregated spirals was markedly dependent on the alkaloid used, on the incubation temperature, and on the absence or presence of MAPs. Aggregated spirals failed to assemble in the presence of high concentrations of MAP-1A or MAP-1B, whereas they assembled readily with tau and MAP-2. Differences in patterns of turbidity development using pure tubulin allowed the classification of thirteen cytotoxic vinca alkaloids into five distinct groups, with centrifugal recovery of aggregated spirals in close agreement with the various turbidity patterns. With microtubule protein, i.e. tubulin preparations containing MAPs, only four groups were defined by turbidity patterns, and centrifugal protein recovery was more divergent. Vinblastine, vincristine, vindesine, and vinorelbine fell into distinct groups under both reaction conditions, and thus they appear to have qualitatively distinguishable in vitro interactions with tubulin. These differential effects on spiral filament and aggregated spiral assembly revealed that the four drugs induce different constraints on the tubulin molecule.