Nucleation Of Aggregates Research Articles

The Role of Interfaces in the Nucleation of Amyloid Nanotubes

The nucleation and growth pathways of cross-s peptide aggregates are widely studied but not fully understood. Using Amyloid-s(16-22), the nucleating core of the Amyloid-s protein, and a rhodamine 110 -labeled peptide (Rh1722) that co-assembles with Amyloid- s(16-22) nanotubes without changing morphology, we investigate the role of interfaces in amyloid nucleation and self assembly. The high sensitivity of the Rhodamine 110 lifetime to its local environment provides a metric for structural heterogeneity, which is exploited in these studies using Fluorescence Lifetime Imaging Microscopy (FLIM). Specifically, we identify the importance of air-solvent and solvent-glass interfaces in the nucleation of cross-s peptide nanostructures and apply FLIM to demonstrate that different interfaces can lead to distinct nucleation and self-assembly pathways and also to differences in the structure of assembled peptides.

Variation of particle size and its distribution during the synthesis of silicalite-1 nanocrystals

Recent studies imply that the external surface area of the nanozeolite product may, at least in some cases, be related to the average size of the particle population participating in aggregative nucleation, a population which itself is a product of aggregation of even smaller primary nanoparticles. This possibility puts more importance on our understanding of the variation of particle size and its distribution during the crystallization of zeolite nanoparticles. Variation of the particle size and PSD during nanoparticle silicalite-1 crystallization was followed with respect to time by a laser light scattering device with a scattering angle of 173°, for several starting synthesis compositions. Effects of varying TPAOH and water contents in the starting synthesis mixtures on the variation with time of the particle sizes and PSDs, especially across the two distinct aggregation events, were investigated. The products were also analyzed by XRD and AFM. Parallel to the decrease in the average particle size of the final product population with increasing alkalinity and organic template content, its PSD was observed to become narrower too. A reversal in the dependence on TPAOH content, of the average size of the population formed by aggregation, with respect to that of the population participating in aggregation, was observed across both aggregation events, implying that smaller particles aggregated to form larger particles, while larger particles aggregated to form smaller particles during these processes, and this was also seen from the AFM images, to be reflected to the surface features of the final product particles.

Mechanisms of Stone Formation.

We have reviewed the general mechanisms involved in kidney stone formation, with reference to those composed of calcium oxalate or phosphate, uric acid, and cystine. These processes include nucleation of individual crystals, aggregation or secondary nucleation to produce small intrarenal multicrystalline aggregates, fixation within the kidney, and further aggregation and secondary nucleation to produce the clinical stone. The factors regulating these processes have been discussed as well as the effects of tubular fluid or urine pH and promoters or inhibitors, including urate or uric acid in the case of calcium oxalate stones, citrate, pyrophosphate, phytate, and urinary proteins. We also discuss the potential for macromolecular inhibitors to actually promote stone formation when they are fixed to some intrarenal structure or if they themselves become aggregated into protein–protein complexes.

Structure, Dynamics and Surface Hydrophobicity of the Cataract-Associated Mutant, Pro23Thr of Human Gamma D-crystallin: Molecular Basis of Cataract Formation

The cataract-associated Pro23Thr (P23T) mutation in human gammaD-crystallin (HGD) is geographically widespread - thus there is considerable interest in determining the molecular basis of opacity. In an earlier study (1), we found that the mutant showed markedly lowered, retrograde solubility compared to wild-type HGD, leading to the conclusion that the aggregation of P23T was mediated by hydrophobic protein-protein interactions. Subsequently, using NMR (2) and a binding assay with the fluorescent dye Bis-ANS (3), we showed that, in fact, hydrophobic patches were generated on the surface of the mutant protein. Those studies (3) also suggested that the binding site for Bis-ANS on P23T may coincide with the self-association site of the mutant and result in its lowered, retrograde solubility. Here we present new NMR-evidence to identify the Bis-ANS binding sites, and using independent NMR dynamics studies, also show that there is a measurable reduction in the flexibility of the peptide backbone near the hydrophobic patches. These two factors, namely the creation of hydrophobic patches and the lowered peptide backbone flexibility, taken together make a compelling argument that the hydrophobic patches may indeed facilitate the nucleation of aggregates of P23T which are held together by net hydrophobic interactions. Such aggregation would explain the retrograde solubility as well as the lens opacity in vivo. 1. Pande et al (2005) Biochemistry, 44, 2491-2500. 2. Pande et al (2009) Biochem. Biophys. Res. Commun., 382, 196-199. 3. Pande et al (2010) Biochemistry,49, 6122-6129. Supported by NIH grants GM085006 to AS and EY010535 to JP.

A Kinetic Aggregation Assay Allowing Selective and Sensitive Amyloid-β Quantification in Cells and Tissues

The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.

Assays for studying nucleated aggregation of polyglutamine proteins

The aggregation of polyglutamine containing protein sequences is implicated in a family of familial neurodegenerative diseases, the expanded CAG repeat diseases. While the cellular aggregation process undoubtedly depends on the flux and local environment of these proteins, their intrinsic physical properties and folding/aggregation propensities must also contribute to their cellular behavior. Here we describe a series of methods for determining mechanistic details of the spontaneous aggregation of polyQ-containing sequences, including the identification and structural examination of aggregation intermediates.

Nucleation Control in the Aggregative Growth of Bismuth Nanocrystals

The kinetics and mechanism of Bi-nanocrystal growth from the precursor Bi[N(SiMe3)2]3 are determined at various Na[N(SiMe3)2] additive concentrations. The results establish that aggregative nucleation and growth processes dominate Bi-nanocrystal formation. The time dependence of the aggregative nucleation rate−the nucleation function−is determined over the range of Na[N(SiMe3)2] concentrations studied. The time width of aggregative nucleation (Δtn) is shown to remain reasonably narrow, and to correlate with the final Bi-nanocrystal size distribution. The maximum aggregative nucleation rate (Γmax) is shown to vary systematically with Na[N(SiMe3)2] concentration, producing a systematic variation in the final nanocrystal mean size. The Na[N(SiMe3)2] additive functions as both a nucleation-control agent and an Ostwald-ripening agent.

One-step synthesis of Ti-MSU and its catalytic performance on phenol hydroxylation

In this study, high-quality Ti-MSU was synthesized by a convenient one-step hydrothermal synthesis procedure. This is the first detailed study on the role of addition time of a Ti source. We found that the addition time of the Ti source can effectively influence the structure and morphology of the product by adjusting the balance of hydrolysis–polymerization and nucleation–aggregation in different self-assembly steps. The optimum synthesized Ti-SMU sample showed a good catalytic performance on the phenol hydroxylation.

Deletion of a Ure2 C-terminal prion-inhibiting region promotes the rate of fibril seed formation and alters interaction with Hsp40

Prions are proteins that can undergo a heritable conformational change to an aggregated amyloid-like state, which is then transmitted to other similar molecules. Ure2, the nitrogen metabolism regulation factor of Saccharomyces cerevisiae, shows prion properties in vivo and forms amyloid fibrils in vitro. Ure2 consists of an N-terminal prion-inducing domain and a C-terminal functional domain. Previous studies have shown that mutations affecting the prion properties of Ure2 are not restricted to the N-terminal prion domain: the deletion of residues 151-158 in the C-domain increases the in vivo prion-inducing propensity of Ure2. Here, we characterized this mutant in vitro and found that the 151-158 deletion has minimal effect on the thermodynamic stability or folding properties of the protein. However, deletion of residues 151-158 accelerates the nucleation, growth and fragmentation of amyloid-like aggregates in vitro, and the aggregates formed are able to seed formation of fibrils of the wild-type protein. In addition, the absence of 151-158 was found to disrupt the inhibitory effect of the Hsp40 chaperone Ydj1 on Ure2 fibril formation. These results suggest that the enhanced in vivo prion-inducing ability of the 151-158 deletion mutant is due to its enhanced ability to generate prion seeds.

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.

Nitroxide-Mediated Surfactant-Free Emulsion Polymerization of n-Butyl Methacrylate with a Small Amount of Styrene

Nitroxide-mediated emulsion polymerization of n-butyl methacrylate (BMA) can produce highly living and well-controlled polymer chains when polymerized in the presence of 10 mol % styrene (St) using a one-pot, differential monomer addition technique. When n-BMA-co-St is polymerized in the presence of a surfactant above the critical micelle concentration, bimodal particle size distributions are obtained, likely as a result of combined micellar and aggregative nucleation mechanisms. This phenomenon is not observed for the more hydrophilic monomer system of methyl methacrylate and styrene. In the absence of surfactant, however, it is possible to prepare stable, monomodal latexes. Using N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl) nitroxide (SG1), we report the first nitroxide-mediated polymerization of n-butyl methacrylate with a small amount of styrene in a facile surfactant-free emulsion polymerization system. The surfactant-free system requires no separate macroinitiator synthesis step and produces highly living polymers with monomodal particle size distributions. The initiator efficiency can be increased by the addition of methyl acrylate or by the addition of surfactant at concentrations below the critical micelle concentration in the absence of methyl acrylate.

Pathway from a Molecular Precursor to Silver Nanoparticles: The Prominent Role of Aggregative Growth

A mechanistic study of Ag-nanoparticle growth by reaction of [(PPh3)2Ag(O2CC13H27)] and AIBN is reported. The half-life for precursor disappearance at 130.0 ± 0.1 °C under the reaction conditions is determined to be 3.65 ± 0.42 min, which defines the time scale for classical (LaMer) nucleation and growth to be within the first 15 min (4 half-lives). The nanoparticle-growth kinetics are separately determined by TEM monitoring and UV−visible spectroscopy. Fits to the kinetic data establish that the active-growth regime extends to 58 min, and that Ostwald ripening ensues shortly thereafter. Evidence for an aggregative nucleation and growth process is obtained. The quantitative data indicate that classical nucleation and growth, aggregative nucleation and growth, and Ostwald ripening occur in consecutive time regimes with little overlap, and that nanoparticle growth is dominated by the aggregative regime. Aggregative growth should be considered a potential contributing mechanism in all nanoparticle-forming re...

Hydrothermal synthesis of ultralong single‐crystalline α‐Ni(OH)2 nanobelts and corresponding porous NiO nanobelts

AbstractUltralong α‐Ni(OH)2 nanobelts with uniform size have been prepared on large scale via a facile template‐free hydrothermal method. The as‐prepared nanobelts were single crystals, with several tens of microns in length and about 100 nm in width. For the whole process, a novel nucleation–aggregation–dissolution–seed‐directed growth mechanism was proposed based on the experimental results. The roles of aqueous ammonia and hydrothermal temperature were also discussed. Furthermore, porous NiO nanobelts were obtained by annealing the as‐prepared Ni(OH)2 nanobelts. This facile, template‐free, and low cost method might feasibly be scaled up for industrial production. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Nucleation Control of Size and Dispersity in Aggregative Nanoparticle Growth. A Study of the Coarsening Kinetics of Thiolate-Capped Gold Nanocrystals

The thermal coarsening (180 °C) of decanethiolate-capped Au nanocrystals is studied at various tetraoctylammonium bromide concentrations. The coarsening kinetics are determined by measuring nanocrystal size distributions (CSDs) as a function of time. The results are shown to be consistent with aggregative nucleation and growth. For each kinetic trial, the time dependence of the aggregative nucleation rate is extracted from the early time CSDs and fitted by a Gaussian profile. The height of the profile is the maximum nucleation rate, Γmax, and the 2σ width is the time window for nucleation, Δtn. These nucleation parameters are shown to control the final mean size and size distribution of the coarsened nanocrystals. The coarsening kinetics are influenced by tetraoctylammonium bromide concentration because the nanocrystals are partially electrostatically stabilized.

From Ultrafine Thiolate-Capped Copper Nanoclusters toward Copper Sulfide Nanodiscs: A Thermally Activated Evolution Route

This report shows that the size, shape, and composition of presynthesized copper nanoparticles can be nanoengineered through exploiting concurrent interparticle aggregative growth and interfacial carbon−sulfur cleavage in a thermally activated evolution route. This is demonstrated by thermally activated processing of ultrafine copper nanoclusters encapsulated with thiolate monolayer (Cun(SR)m) toward semiconducting copper sulfide (Cu2S) nanodiscs with controllable sizes and shapes. Under controlled temperatures (120−150 °C), the ultrafine Cun(SR)m nanoclusters, with a size of ∼0.5 nm evidenced by TEM, SAXS-WAXS, DCP-AES, and MALDI-TOF measurements, were shown to evolve into thiolate-capped Cu2S nanodiscs via thermally activated coalescence and copper-catalyzed interfacial C−S cleavage reactivities. The Cu2S nanodiscs, as confirmed by XPS and HRTEM analyses, exhibited controllable and monodispersed sizes depending on the thermal processing parameters, ranging from 5 to 35 nm in the disk dimension and 3−6 nm in the thickness dimension. These nanodiscs are stable and display remarkable 1D/2D ordering upon self-assembly. This process is not a simple digestive ripening of smaller particles because it involves an aggregative nucleation and growth process distinctively different from traditional ripening and a reactive carbon−sulfur bond cleavage controlled by the catalytic effect of copper under the specified temperatures. The coupling of the thermally activated coalescence and C−S bond cleavage to convert the ultrafine Cu nanoclusters toward the formation of Cu2S nanodiscs is highly effective for tuning nanoscale size, shape, and composition, and could find applications in nanoengineering a variety of semiconducting nanocrystals for applications in nanostructured electronic, sensing, and photochemical devices.

Characterization of High-Molecular-Weight Nonnative Aggregates and Aggregation Kinetics by Size Exclusion Chromatography With Inline Multi-Angle Laser Light Scattering

Size exclusion chromatography with an inline multi-angle light scattering detector (SEC-MALS) was assessed as a means to characterize and monitor the formation of soluble, high-molecular-weight (HMW) protein aggregates so as to better quantify and model nonnative aggregation kinetics. Assay configuration and robustness were tested with respect to sample preparation, column type, and assay variability. Independent comparison of SEC-MALS with batch light scattering analysis indicates good agreement between the two methods. Weight-average molecular weight (Mw), radius of gyration (Rg), apparent polydispersity, and mass fraction monomer (m) together are shown to provide qualitative and quantitative experimental signatures to distinguish high-MW aggregate growth via chain polymerization versus that via aggregate–aggregate condensation. Mechanistic treatment of aggregation kinetics monitored by SEC-MALS is illustrated by data regression using a recently developed Lumry-Eyring Nucleated Polymerization model that explicitly accounts for aggregate nucleation and competing growth via chain- and condensation-polymerization. The combination of time-dependent Mw and m data are shown to provide a convenient and robust means to separate and quantify characteristic time scales or rate coefficients for concurrent stages of irreversible aggregation. In addition, the scaling of Rg with Mw for HMW aggregates provides useful insights into aggregate morphology and mechanisms of aggregate growth. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3997–4016, 2009

Size sorting of citrate reduced gold nanoparticles by sedimentation field-flow fractionation

Gold nanoparticles (GNPs) have been synthesized through the citrate reduction method; the citrate/gold(III) ratio was changed from 1:1 up to 10:1 and the size of the resulting nanoparticles was measured by sedimentation field-flow fractionation (SdFFF). Experimental data showed that the GNPs size decreases in the ratio range 1:1–3:1 and then increases from 5:1 to 10:1 passing through a plateau region in between, and is almost independent of the precursor solution concentrations. In the zone of minimum diameters the synthetic process does not produce monodispersed GNPs but often multiple distributions, very close in size, are observed as evidenced by the particle size distributions (PSDs) derived from the SdFFF fractograms. UV–vis spectrophotometry, being the most common technique employed in the optical characterization of nanoparticles suspensions, was used throughout this work. A confirmation of the nucleation–aggregation–fragmentation mechanism was inferred from the cross-correlation between UV–vis and SdFFF results.

Supported Lipid Bilayer Templated J-Aggregate Growth: Role of Stabilizing Cation−π Interactions and Headgroup Packing

Controlling the self-assembly of molecules into specific structural motifs has important implications for the design of materials with specific optical properties. We report here the results of a correlated confocal fluorescence-atomic force microscopy (AFM) study of pseudoisocyanine iodide (PIC) self-assembly on supported lipid bilayers. Through judicious selection of bilayer headgroup packing and chemistry, two types of PIC J-aggregates, distinguishable by their absorbance spectra, and both exhibiting strong resonant fluorescence and bathochromic shifts in absorbance relative to the monomer, were isolated. Remarkably, selective templating can be achieved using different zwitterionic headgroups, producing J-aggregates that display a larger bathochromic shift than their solution counterparts. Our correlated confocal-AFM studies coupled with FT-IR spectroscopy suggested that zwitterionic phospholipids mediate J-aggregate formation through specific cation-pi interactions between PIC and the lipid headgroups with the PIC molecules oriented largely perpendicular to the bilayer normal. The existence of the two isoforms further suggests that bilayer headgroup packing plays a key role in controlling interchromophore organization and subsequent aggregate nucleation and growth.

Surfactant-free fabrication of α-Fe 2O 3 structures with flower-like morphology in aqueous solution

Monodisperse hematite (α-Fe 2O 3) nanostructures with flower-like morphology were obtained by a simple forced hydrolysis and thermal decomposition method without the assistance of any template or organic surfactant. The microstructure of the final flower-like particles was characterized in detail by transmission electron microscopy and selected area electron diffraction, which showed the composite structure of the particle “core”. Electron diffraction patterns obtained for sections located on the “leaf” of the particles were consistent with hematite single crystals, whereas those obtained for the middle of the particle showed additional spots, due to the presence of goethite in the particle core. Fourier transform infrared spectroscopy and X-ray diffraction also confirmed the presence of goethite phase in the particle. A mechanism based on nucleation–aggregation–recrystallization, which speculated that the goethite precipitates first and then hematite heterogeneously nucleates on goethite particles, was proposed.

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.