Short Oligomers Research Articles

Evidence of Direct Binding of G-Actin and Calmodulin to PMCA by Surface Plasmon Resonance

Previous studies of our laboratory suggest that the main isoform of the plasma membrane calcium ATPase (PMCA) from erythrocytes, PMCAh4b, associates with the actin cytoskeleton and its catalytic activity can be regulated through this interaction. Apparently, G-actin and/or short oligomers enhance the catalytic activity of the pump, while F-actin seems to inhibit it. To further explore the interaction between PMCA and G-actin, we performed binding experiments based on Surface Plasmon Resonance technology (SPR) using purified PMCAh4b from erythrocytes and purified rabbit muscle G-actin on a BIAcore T100 system. Our strategy consisted in immobilizing G-actin to the sensor chip surface while PMCA constituted the analyte (125 nM to 2 μM). The sensorgrams obtained showed an increase in the response as a function of PMCA concentration with a saturable binding. The unspecific binding values were subtracted from the response obtained in the control flow cell (without immobilized G-actin). The apparent KD (1.25 μM) was determined by kinetic analysis with parameters kon (1.33±0.07) 104 M-1s-1 and koff (1.66±0.03) 10-2 s-1.Conversely, we immobilized PMCAh4b to the sensor chip surface. The analytes were (i) G-actin (0.6 to 5 μM) or (ii) Calmodulin, a known activator of PMCAh4b (1.8 to 15 nM). Results show that G-actin binds to the activated sensor with KD=3μM, kon (2.98±0.45) 104 M-1s-1 and koff (8.98±0.67) 10-2 s-1 and calmodulin binds with an apparent KD of (1.14±0.6) 10-8 M. This last value agrees with the known value of calmodulin binding affinity determined by other methods.These results show a specific and high apparent affinity binding between PMCA and G-actin and reveal the usefulness of SPR technology to determine binding constants between small molecules and a large membrane protein like the plasmatic calcium pump. With grants of ANPCYT/CONICET/UBACYT.

Neutral Nickel Oligo- and Polymerization Catalysts: The Importance of Alkyl Phosphine Intermediates in Chain Termination

An unconventional chain termination reaction has been explored for the SHOP (Shell higher olefin process)-type, anilinotropone, and salicylaldiminato nickel-based oligo- and polymerization catalysts by using density functional theory (DFT). Starting from the tetracoordinate alkyl phosphine complex, the termination reaction was found to involve a rearrangement of the alkyl chain to form a pentacoordinate β-agostic complex, β-hydride elimination, and olefinic chain dissociation and to compete with propagation at sufficiently high phosphine concentration and/or basicity. It provides the first complete and convincing mechanistic rationale for the decreasing chain lengths observed upon increasing phosphine concentration and basicity. The unconventional reaction was found to be a major termination pathway for the SHOP-type catalyst and is very unlikely to lead to branching and olefin isomerization, which is critical for explaining why the SHOP catalyst, in contrast to the anilinotropone and salicylaldiminato catalysts, tends to lead to the oligomerization of ethylene to form linear α-olefins. Based on our results we have proposed a new and extended catalytic cycle for the SHOP-type ethylene oligomerization catalyst. Finally, the importance of the new termination reaction for the SHOP-type catalyst suggests that this reaction may also operate with other ethylene oligomerization nickel catalysts. This prediction was confirmed for a pyrazolonatophosphine catalyst, for which the new termination route was found to be even more facile, which explains the short oligomers produced by this catalyst.

DNA Binding Compatibility of the Streptococcus pneumoniae SsbA and SsbB Proteins

Background Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated.Methodology/Principal FindingsThe complexes that were formed by the SsbA and SsbB proteins on dTn oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dTn oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dTn oligomers via a “shared-strand binding” mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins.Conclusion/SignificanceThese results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.

On the Structure and Stretching of Microhydrated DNA

The structure of short double-stranded DNA oligomers in complex with varied amounts of water is investigated with classical molecular dynamics. Free simulations are performed first and mechanical stress is switched on afterward, resembling the conditions of single-molecule conductivity experiments. Water as well as counterions are seen to contribute to the stabilization of double-stranded DNA structure, and a collapse of the native DNA structure is observed upon the removal of a certain amount of water. Pulling with a moderate external force provides additional support to the double-stranded DNA structure, whereas larger forces lead to the overstretching transition followed by the separation of DNA strands, in a manner similar to that observed in fully hydrated DNA.

Hydrodynamic Properties of Wormlike Macromolecules: Monte Carlo Simulation and Global Analysis of Experimental Data

A Monte Carlo simulation, coupled with bead-model hydrodynamic calculation, has been employed to predict hydrodynamic coefficients and other solution properties, of wormlike macromolecules, covering the full range of the wormlike model, from short cylinders to very long, fully flexible chains, eventually including excluded-volume effects. The results have been implemented in a computational tool, Multi-HYDFIT, which performs the determination of the structural parameters from a set of experimental data of various properties for multiple samples with varying molecular weight. An analysis of experimental data of double-stranded DNA demonstrates that the Multi-HYDFIT treatment, with our simulation results, predicts the various solution properties of DNA in an extremely wide range of sizes, from 8 to 200 000 base pairs, yielding values of the parameters that agree with those of the double helix. The scheme is also applied to other synthetic and biological macromolecules, like the very stiff, triple-helical schizophyllan polysaccharide or the very flexible poly(isobutylene) polymer, the latter covering again an extremely wide range that includes quite short oligomers.

Isozymes from the herbivorous gecarcinid land crab, Gecarcoidea natalis that possess both lichenase and endo-β-1,4-glucanase activity

Three isozymes with both lichenase and endo-β-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-β-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-β-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked β-1,4-glucosidase activity as they could not hydrolyse cellobiose.

Density functional theory study of the structure and energetics of negatively charged oligopyrroles

First-principles calculations are used to investigate the electronic properties of negatively charged n-pyrrole oligomers with n = 2–18. Chains of neutral oligomers are bent, whereas the negatively charged oligomers become almost planar due to accumulation of negative charge at the end monomers. Isomers of short oligomers (n < 6) display negative electron affinity although the corresponding anions are energetically stable. For longer oligomers with n ≥ 6, the electron affinity is small and positive, slowly increasing with oligopyrrole length. Doping of 12-pyrrole with lithium atoms shows that negative oxidation states are possible due to electron transfer from dopant to oligomer at locations close to dopant. These 12-pyrrole regions support extra negative charge and exhibit a local structural change from benzenoid to quinoid structure in the CC backbone conjugation. Comparison between neutral and doped polypyrrole (PPy) indicates that doped polymers displays a substantial depletion of the band gap energy and the appearance of dopant-based bands in the gap for a 50% per monomer doping level. It is predicted that Li-doped PPy is not metallic. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

A potential control point of glucose delivery from starchy foods: intestinal mucosal α‐glucosidase digestion

To control the glucose delivery from starchy foods, much attention has been given to α‐amylase (AMY), as opposed to the contribution from mucosal α‐glucosidases. The aim of this research was to investigate the digestion capability of mucosal α‐glucosidases on various starch structures to explore their possible expanded roles in digestion. Substrates were incubated with recombinant N‐ (Nt) and C‐terminals (Ct) of maltase‐glucoamylase (MGAM) and sucrase‐isomaltase (SI) for different digestion periods, and the glucogenesis and residue structures were examined. At raw starch granule level, mucosal α‐glucosidases showed the capability to produce glucose from uncooked starch granules at a low digestion completion. At the cooked starch level, CtMGAM showed high digestion completion near 80% without AMY participation; other three subunits reached 20–30% digestion. At the α‐limit dextrin (LDx) level, the four subunits showed different roles where NtMGAM only digested short linear oligomers but the other three subunits individually digested large branched molecules with different completion percentage. Collectively, these findings suggest mucosal α‐glucosidases, particularly CtMGAM, may be potential control points for glucose delivery in the human body. This work was supported by internal funding from the Whistler Center and Children's Nutrition Research Center of the ARS/USDA.

An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Identification and Quantification of Short Oligomeric Proanthocyanidins and Other Polyphenols in Boysenberry Seeds and Juice

Proanthocyanidins and other polyphenols in the seeds and juice of boysenberry were quantitatively analyzed. Polyphenolic extracts were prepared from the waste seeds and commercial juice by chromatographic fractionation. Compositional analysis revealed that both extracts contained six polyphenolic classes: flavanol monomers, proanthocyanidins, anthocyanins, ellagic acid, ellagitannins, and flavonol glycosides. Ellagitannins were the most abundant polyphenols in both extracts. Proanthocyanidins were present as short oligomers consisting of dimeric and trimeric procyanidins and propelargonidins, with the most abundant component being procyanidin B4 in both extracts. Quantification by high-performance liquid chromatography-mass spectrometry (HPLC-MS) revealed that the seeds contained a 72-fold higher amount of proanthocyanidins than the juice. These results indicate that boysenberry fruits contain short oligomeric proanthocyanidins along with flavanol monomers and the seeds represent a good source of short oligomeric proanthocyanidins.

Highly Sulphonated Multiblock‐co‐polymers for Direct Methanol Fuel Cells

AbstractMultiblock‐co‐polymers with different ion exchange capacities were synthesised by nucleophilic aromatic step‐growth polycondensation of hydrophilic and hydrophobic oligomers. The main focus of this work was to investigate the dependence of oligomer reactivity on molecular weight and how the reaction conditions need to be changed to obtain high‐molecular multiblock‐co‐polymers, consisting of long block segments. Multiblock‐co‐polymers composed of short oligomers could be synthesised under mild reaction conditions. The coupling of longer oligomers required more basic reaction conditions, because the reactive functional end‐groups were shielded by the long oligomeric main chain. The membranes were investigated further in terms of their properties relevant for use as polymer electrolyte membrane in direct methanol fuel cells (DMFCs). The mechanical stability of the acidic polymers was improved through ionical crosslinking with basic polybenzimidazole. A series of membranes was tested in a self‐ and in an air‐breathing DMFC.

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670nm. We developed a new approach to antibody conjugation, using Huisgen-Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.

The adsorption of short single-stranded DNA oligomers to mineral surfaces

We studied the adsorption of short single-stranded deoxyribonucleic acid (ssDNA) oligomers, of approximately 30 nucleotides (nt) in length, of varying sequence, adenine + guanine + cytosine (AGC) content, and propensity to form secondary structure, to equal surface area samples of olivine, pyrite, calcite, hematite, and rutile in 0.1 M NaCl, 0.05 M pH 8.1 KHCO 3 buffer. Although the mineral surfaces have widely varying points of zero charge, under these conditions they show remarkably similar adsorption of ssDNA regardless of oligomer characteristics. Mineral surfaces appear to accommodate ssDNA comparably, or ssDNA oligomers of this length are able to find binding sites of comparable strength and density due to their flexibility, despite the disparate surface properties of the different minerals. This may partially be due charge shielding by the ionic strength of the solutions tested, which are typical of many natural environments. These results may have some bearing on the adsorption and accumulation of biologically derived nucleic acids in sediments as well as the abiotic synthesis of nucleic acids before the origin of life.

Microsecond Single-Molecule Tracking ( μsSMT)

Here we report on a method to track individual molecules on nanometer length and microsecond timescales using an optical microscope. Our method is based on double-labeling of a molecule with two spectrally distinct fluorophores and illuminating it with laser pulses of different wavelengths that partially overlap temporally. We demonstrate our method by using it to resolve the motion of short DNA oligomers in solution down to a timescale of 100 μs.

Chain Length Dependence in Diketopyrrolopyrrole/Thiophene Oligomers

Diketopyrrolopyrrole-based polymers have shown good performance in polymer solar cells. Monodisperse short oligomers, monomer up to tetramer, of one of these polymers, pBBTDPP1, have been prepared and the chain length dependence of their optical and electrochemical properties has been investigated. The optical and electrochemical data obtained in solution lead to the conclusion that conjugation in this system is very limited. The most probable reason for this is a twist in the backbone, caused by steric hindrance of the dodecyl side chains with the sulfur atoms. In thin films, the chain length dependence of the optical properties is much stronger, due to planarization of the oligomer chains upon aggregation.

A neutron reflectivity study of the interfacial and thermal behaviour of surface-attached hairpin DNA

Mixed self-assembled monolayers (mSAMs) have been successfully utilised as platforms for gene sensors, employing optical as well as electrochemical means of detection. Probe density is one of the most important parameters in the construction of such a sensor and thus a fundamental understanding of the structure within the mSAM is vital. In this work, the interfacial behaviour of mixed SAMs, where short PEG oligomers co-adsorbed to the surface with hairpin structured oligonucleotide (ODN) probes, has been investigated. The neutron reflectivity of the mixed SAMs was measured at differing HPP : PEG ratios, and through two routes of formation, to elucidate the effect of controlled HPP surface density on surface conformation of the probes and on the final hybridised ODN–HPP construct. General conclusions regarding the structure of the investigated SAMs could be drawn from determined thickness and volume fraction values and conformational changes in the mSAM, induced by hybridisation with complementary ODN, were also detected. An investigation of the melting behaviour of the surface-attached HPPs was also conducted with polarised neutron reflectivity and clear signs of melting were observed in the reflectivity and the SLD profiles around 45 °C.

Dielectrophoretic tweezers as a platform for molecular force spectroscopy in a highly parallel format.

We demonstrated the application of a simple electrode geometry for dielectrophoresis (DEP) on colloidal probes as a form of molecular force spectroscopy in a highly parallel format. The electric field between parallel plates is perturbed with dielectric microstructures, generating uniform DEP forces on colloidal probes in the range of several hundred piconewtons across a macroscopic sample area. We determined the approximate crossover frequency between negative and positive DEP using electrodes without dielectric microstructures-a simplification over standard experimental methods involving quadrupoles or optical trapping. 2D and 3D simulations of the electric field distributions validated the experimental behavior of several of our DEP tweezers geometries and provided insight into potential improvements. We applied the DEP tweezers to the stretching of a short DNA oligomer and detected its extension using total-internal reflection fluorescence microscopy. The combination of a simple cell fabrication, a uniform distribution of high axial forces, and a facile optical detection of our DEP tweezers makes this form of molecular force spectroscopy ideal for highly parallel detection of stretching or unbinding kinetics of biomolecules.

Comparison of methods for orienting and aligning DNA origami

DNA origami are self-assembling units that can themselves undergo further self-assembly to form oligomers. These oligomers have potential applications for assembly of nanoelectronic or nanophotonic circuitry or for assembly of biological components. However, there are a variety of assembly defects that must be controlled. These include defects in alignment between the origami (origami that are offset from each other), defects in relative orientation (origami that are rotated relative to one another), and defects in up/down orientation (neighboring origami that are not both face up or face down). Four strategies for controlling the oligomerization of DNA origami were compared. We tested (i) inclusion of T-bumpers, (ii) omission of staple strands on the edge of the DNA origami, (iii) varying numbers and lengths of single-stranded linkers and the stoichiometry of linking strands, and (iv) optimization of annealing time and temperature. The DNA origami chains were characterized both on mica and cationic SAMs on silicon [100] by tapping mode AFM in the air. Orientations were verified by observation of loop regions on the DNA origami and an intentionally designed notch that makes the origami structure chiral on the surface. AFM images showed that inclusion of T-bumpers failed to block π-stacking interactions, and that inclusion of single stranded linkers greatly reduced alignment defects but did not control defects in relative orientation. On the other hand, single stranded linkers in conjunction with elimination of staple strands from the edge of the DNA origami formed short oligomers with good alignment and good relative orientation. We investigated the optimal annealing temperature and time with quenching experiments and observed stepwise origami formation and oligomerization.

Peptoid origins

Peptoid oligomers were initially developed as part of a larger basic research effort to accelerate the drug-discovery process in the biotech/biopharma industry. Their ease of synthesis, stability, and structural similarity to polypeptides made them ideal candidates for the combinatorial discovery of novel peptidomimetic drug candidates. Diverse libraries of short peptoid oligomers provided one of the first demonstrations in the mid-1990s that high-affinity ligands to pharmaceutically relevant receptors could be discovered from combinatorial libraries of synthetic compounds. The solid-phase submonomer method of peptoid synthesis was so efficient and general that it soon became possible to explore the properties of longer polypeptoid chains in a variety of areas beyond drug discovery (e.g., diagnostics, drug delivery, and materials science). Exploration into protein-mimetic materials soon followed, with the fundamental goal of folding a non-natural sequence-specific heteropolymer into defined secondary or tertiary structures. This effort first yielded the peptoid helix and much later the peptoid sheet, both of which are secondary-structure mimetics that are close relatives to their natural counterparts. These crucial discoveries have brought us closer to building proteinlike structure and function from a non-natural polymer and have provided great insight into the rules governing polymer and protein folding. The accessibility of peptoid synthesis to chemists and nonchemists alike, along with a lack of information-rich non-natural polymers available to study, has led to a rapid growth in the field of peptoid science by many new investigators. This work provides an overview of the initial discovery and early developments in the peptoid field.

Differentiating a Diverse Range of Volatile Organic Compounds with Polyfluorophore Sensors Built on a DNA Scaffold

Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which DNA bases are replaced with fluorophores. A preliminary study reported that some sequences of ODFs were able to respond to a few organic small molecules in the vapor phase, giving a change in fluorescence. Here, we follow up on this finding by investigating a larger range of volatile organic analytes, and a considerably larger set of sensors. A library of tetramer ODFs of 2401 different sequences was prepared by using combinatorial methods, and was screened in air for fluorescence responses to a set of ten different volatile organics, including multiple aromatic and aliphatic compounds, acids and bases, varied functional groups, and closely related structures. Nineteen responding sensors were selected and characterized. These sensors were cross-screened against all ten analytes, and responses were measured qualitatively (by changes in color and intensity) and quantitatively (by measuring ΔR, ΔG, and ΔB values averaged over five to six sensor beads; R=red, G=green, B=blue). The results show that sensor responses were diverse, with a single sensor responding differently to as many as eight of the ten analytes; multiple classes of responses were seen, including quenching, lighting-up, and varied shifts in wavelength. Responses were strong, with raw ΔR, ΔG, and ΔB values of as high as >200 on a 256-unit scale and unamplified changes in many cases apparent to the naked eye. Sensors were identified that could distinguish clearly between even very closely related compounds such as acrolein and acrylonitrile. Statistical methods were applied to select a small set of four sensors that, as a pattern response, could distinguish between all ten analytes with high confidence. Sequence analysis of the full set of sensors suggested that sequence/order of the monomer components, and not merely composition, was highly important in the responses.