Oligomers In Solution Research Articles

Mechanical Stirring Induces Heteroaggregate Formation and Self‐assembly of Pseudoenantiomeric Oxymethylene Helicene Oligomers in Solution

AbstractA mixture of pseudoenantiomeric oxymethylene helicene (P)‐pentamers and (M)‐hexamers formed heteroaggregates and self‐assembled as a result of mechanical stirring in solution at 25 °C. No aggregation occurred without stirring, and heteroaggregation was accelerated at high stirring rates. Repeatedly alternating between mechanical stirring and stopping respectively induced and then ceased the heteroaggregation. Sonication also promoted the heteroaggregation, which did not occur at high pressures or during centrifugation. Mechanisms based on friction due to mechanical stirring, which generated local and temporal high‐temperature domains, are considered. It is shown that different methods of mechanical stimulation, namely, stirring and surface contact, induce enantiomeric heteroaggregate formation.

Murine ultrasound-guided transabdominal para-aortic injections of self-assembling type I collagen oligomers

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.

Aggregation and Disaggregation of Morpholino Oligomers in Solution.

Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.

Resin adhesion strengths to zirconia ceramics after primer treatment with silane coupling monomer or oligomer.

Resin bonding to zirconia ceramics is difficult to achieve using the standard methods for conventional silica-based dental ceramics, which employ silane coupling monomers as primers. The hypothesis in this study was that a silane coupling oligomer -a condensed product of silane coupling monomers- would be a more suitable primer for zirconia. To prove this hypothesis, the shear bond strengths between a composite resin and zirconia were compared after applying either a silane coupling monomer or oligomer. The shear bond strength increased after applying a non-activated ethanol solution of the silane coupling oligomer compared with that achieved when applying the monomer. Thermal treatment of the zirconia at 110°C after application of the silane coupling agents was essential to improve the shear bond strength between the composite resin cement and zirconia.

Insight into the oligomeric structure of PORA from A. thaliana

Light-dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a plant enzyme that directly needs light to conduct a biochemical reaction. In the present paper we confirmed that POR forms large oligomers in solution before binding of substrates. We carried out the research using different techniques: cross-linking, native gel electrophoresis and FRET measurements. Mass spectrometry analysis of the cross-link products provided the first structural data about the organisation of the oligomer of POR. The results indicated that the catalytic motifs of the adjacent subunits become close to each other upon binding of substrates. Moreover, we identified two mutations of POR that disturbed its oligomerisation properties: Δ85–88 and Δ240–270. Additionally, a complete loss of the catalytic activity was observed for the following mutations: Δ189–194, Δ240–270, Δ318–331 and Δ392–393.

Nanoprecipitation–Miniemulsion polymerization combined method: A novel approach to synthesis drug loaded nanoparticles with tunable characteristics

In this research polystyrene nanoparticles (NPs) were synthesized using a novel straightforward approach based on the nanoprecipitation and miniemulsion polymerization. Nanoprecipitation or solvent shifting method was applied to prepare primary emulsions. For this purpose, oil phase consisting of solution of styrene, alcohol and polylactide oligomer as a cosurfactant, was added to aqueous phase consisting of water and surfactant. Primary emulsions were easily formed as a result of water-alcohol interdiffusion. Afterwards polystyrene NPs were synthesized by free radical miniemulsion polymerization process. Effect of variety of parameters, such as initiator nature (water-soluble or oil-soluble), cosurfactant, styrene to alcohol and alcohol to water ratio, on polymerization yield and NPs characteristics were investigated. Sodium 11-acryloyl-aminoundecanoate was tested as an amphiphilic polymerizable surfactant to synthesize polystyrene NPs with surface functional groups. Moreover, to examine the applicability of proposed approach for drug immobilization, rifampicin as a hydrophobic anti tubercular model drug was investigated. NPs characteristics were analyzed using scanning electron microscopy, light scattering and zeta potential measurements. Consequently, it was shown that nanoprecipitation–miniemulsion polymerization approach can be considered as a straightforward, low energy emulsification method to synthesize polymeric NPs with tunable characteristics.

Density functional theory of freezing of a system of highly elongated ellipsoidal oligomer solutions

ABSTRACTWe have used the density functional theory of freezing to study the liquid crystalline phase behavior of a system of highly elongated ellipsoidal conjugated oligomers dispersed in three different solvents namely chloroform, toluene and their equimolar mixture. The molecules are assumed to interact via solvent-implicit coarse-grained Gay–Berne potential. Pair correlation functions needed as input in the density functional theory have been calculated using the Percus–Yevick (PY) integral equation theory. Considering the isotropic and nematic phases, we have calculated the isotropic–nematic phase transition parameters and presented the temperature–density and pressure–temperature phase diagrams. Different solvent conditions are found not only to affect the transition parameters but also determine the capability of oligomers to form nematic phase in various thermodynamic conditions. In principle, our results are verifiable through computer simulations.

Separation and Purification of Furfuryl Alcohol Monomer and Oligomers Using a Two-Phase Extracting Process

Aqueous two-phase extraction processes were applied for the first time for the separation and purification of furfuryl alcohol monomer and oligomers. Deionized water was used as the liquid–liquid extraction solvent whereas magnesium sulfate acted as the salting-out reagent. Furfuryl alcohol preferentially partitioned to the water phase and could be further extracted from its aqueous solution due to the decreased solubility in salt rich phase. Various influences, such as partition coefficient and extractability, were studied during the liquid–liquid extraction. The extraction by using deionized water resulted in a high oligomer content around ∼94 wt % in the separated furfuryl alcohol oligomer solution whereas the salting-out furfuryl alcohol showed a purity of ∼92 wt %.

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides.

The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.

HCN, Formamidic Acid, and Formamide in Aqueous Solution: A Free-Energy Map.

What chemical species might be found if water or ammonia reacts with HCN in aqueous solution under neutral conditions? Is it energetically favorable for formamidic acid, the first hydration product of HCN, to tautomerize into formamide under standard conditions? Do these molecules form stable oligomers in solution? To answer these questions, we constructed a Gibbs free-energy map of the molecules that might be present to evaluate their relative thermodynamic and kinetic stability. Our protocol utilizes density functional theory calculations, Poisson-Boltzmann implicit solvent, and thermodynamic corrections. We find that for C1 species, formamide is indeed the thermodynamic sink, although the initial barrier to hydration is ∼30 kcal/mol. Molecules with one carbon and three heteroatoms are less stable. We also find that for HCN trimerization, although the planar sp(2) six-membered ring is more stable compared to its monomers, the reverse is true for the nonplanar sp(3) six-membered rings formed by trimerization of formamidic acid or formamide.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A-C16A mutant did not formed oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A-C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway?

Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression of nontoxic amylin monomers into cytotoxic oligomers are not very clear. Studies based on Amyloid-β (Aβ), a peptide with similar size, have suggested that a conformational transition along the aggregation pathway makes the oligomers of Aβ membrane-binding competent (1). We have explored the possibility of amylin undergoing a similar transition during aggregation as it could shed light on potential commonalities in the conformation and function of different toxic amyloid species. Using fluorescence correlation spectroscopy, we identify two distinct oligomers of amylin along the aggregation pathway having hydrodynamic radius of 0.90 nm and 1.6 nm respectively. The membrane affinity of amylin increases remarkably from ∼15 % in smaller species to ∼ 85% in the larger one as assessed by an in vitro membrane-binding assay developed in our lab (2). We observe similar difference in the cell membrane attachment ability of these two species in RIN5mf cell lines using confocal microscopy. A preliminary conformational study in artificial lipid bilayers using a SERS based methodology (3) suggests a temporal conformational reorganization in the peptide backbone. Our data suggest that amylin might acquire toxic function by a mechanism which depends on similar conformational features as Aβ in presence of membranes. Further studies aimed at obtaining high-resolution structural details of amylin oligomers in solution and in membranes are currently in progress.

Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis

Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg2+ ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions.

A Kinetic Monte Carlo Simulation Study of Synthesis Variables and Diffusion Coefficients in Early Stages of Silicate Oligomerization

A kinetic Monte Carlo (kMC) approach combined with density functional theory (DFT) calculations is used to examine the effects of molecular diffusion and synthesis parameters (pH 7–12) as well as initial monomer concentration (0.01–1 mol L–1) for a silicate oligomerization model. To implement this approach, we have adapted the open source kMC SPPARKS software in order to simulate the early stages involved in zeolite formation and more generally kinetically driven reactional systems, using a variety of lattice models (fcc/octahedral/tetrahedral, i.e., fcc/oct/tet). First, these adaptations were validated with kinetically driven reactional systems of the “Lodka” model, providing an excellent match with the analytical solution of the reactive system. The calculations reveal that both the lattice complexity and the diffusion coefficients of species have an impact on the steady state concentration (ssc) of oligomers in solution. Second, the approach is further applied to the early stages of silicate oligomeriz...

Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA

The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and –C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA.

Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations.

Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimer's disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aβ oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aβ, FRET-FCS detected oligomers consisting of less than 10 Aβ molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aβ oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Ludwig-Soret effect of aqueous solutions of ethylene glycol oligomers, crown ethers, and glycerol: Temperature, molecular weight, and hydrogen bond effect.

The thermal diffusion, also called the Ludwig-Soret effect, of aqueous solutions of ethylene glycol oligomers, crown ethers, and glycerol is investigated as a function of temperature by thermal diffusion forced Rayleigh scattering. The Soret coefficient, ST, and the thermal diffusion coefficient, DT, show a linear temperature dependence for all studied compounds in the investigated temperature range. The magnitudes and the slopes of ST and DT vary with the chemical structure of the solute molecules. All studied molecules contain ether and/or hydroxyl groups, which can act as acceptor or donor to form hydrogen bonds, respectively. By introducing the number of donor and acceptor sites of each solute molecule, we can express their hydrogen bond capability. ST and DT can be described by an empirical equation depending on the difference of donor minus acceptor sites and the molecular weight of the solute molecule.

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers.

Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures.

The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are able to protect and recondition the cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP20–29 fragment or by a low potassium medium, regardless of their capacity for accelerating or inhibiting in vitro formation of fibers. In vivo animal experiments are required to study the impact of chemical chaperones in cognitive and metabolic syndromes.