Nucleated Polymerization Research Articles

Controlling the Cooperativity in the Supramolecular Polymerization of Ionic Discotic Amphiphiles via Electrostatic Screening.

In a combined experimental and theoretical approach, we investigate the supramolecular polymerization of ionic discotic amphiphiles into nanorods of varying mean length, depending on the temperature and ionic strength of the buffered aqueous solution. Invoking a nucleated supramolecular polymerization model that explicitly deals with the effects of screened Coulomb interactions, we correlate the degree of cooperativity of the supramolecular polymerization with the ionic strength of the solution, as probed by means of circular dichroism spectroscopy. Experiment and theory show that electrostatic interactions between the amphiphiles in the rods make the polymerization less cooperative, implying that the larger the concentration of mobile ions in the solution the larger the cooperativity due to their screening effect. We furthermore extract quantitative information about the effective surface charge densities of the supramolecular nanorods in solution, a parameter that has been particularly difficult to determine experimentally in other related self-assembled systems.

Scanning electrochemical microscopy and video microscopy investigations of Tiron-mediated polypyrrole nucleation on AA2024-T3

Scanning electrochemical microscopy (SECM) and video microscopy have been used to examine the mediated electrodeposition of polypyrrole on AA2024-T3. Of particular interest is the role of surface heterogeneity (namely, copper-rich secondary phase particles) on electrodeposition mediated by 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). SECM shows that polymer nucleation occurs exclusively on the aluminum matrix of the alloy. Video microscopy shows this to be true on a model alloy (a pure Al substrate with an embedded Cu wire) as well, and also suggests that polymer growth is directional toward the copper-rich sites in the absence of sulfate in the deposition solution. A model is presented in which polymer deposition on the copper-rich sites is inhibited either by CuSO4-induced passivation or by the loss of mediator due to Cu–Tiron complex formation.

Efficiency of organelle capture by microtubules as a function of centrosome nucleation capacity: general theory and the special case of polyspermia.

Transport of organelles along microtubules is essential for the cell metabolism and morphogenesis. The presented analysis derives the probability that an organelle of a given size comes in contact with the microtubule aster. The question is asked how this measure of functionality of the microtubule aster is controlled by the centrosome. A quantitative model is developed to address this question. It is shown that for the given set of cellular parameters, such as size and total tubulin content, a centrosome nucleation capacity exists that maximizes the probability of the organelle capture. The developed general model is then applied to the capture of the female pronucleus by microtubules assembled on the sperm centrosome, following physiologically polyspermic fertilization. This application highlights an unintuitive reflection of nonlinearity of the nucleated polymerization of the cellular pool of tubulin. The prediction that the sperm centrosome should lower its nucleation capacity in the face of the competition from the other sperm is a stark illustration of the new optimality principle. Overall, the model calls attention to the capabilities of the centrosomal pathway of regulation of the transport-related functionality of the microtubule cytoskeleton. It establishes a quantitative and conceptual framework that can guide experiment design and interpretation.

Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive antiparallel β-sheet structure

Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.

RAFT-mediated emulsion polymerization of vinyl acetate: a challenge towards producing high molecular weight poly(vinyl acetate)

The preparation of poly(vinyl acetate) with well-controlled structure has received a great deal of interest in recent years because of a large number of developments in living radical polymerization techniques. Among these techniques, the use of reversible addition–fragmentation chain transfer (RAFT)-mediated polymerization has been employed for the controlled polymerization of vinyl acetate due to the high susceptibility of this monomer towards chain transfer reactions. Here, a novel water-soluble N,N-dialkyl dithiocarbamate RAFT agent has been prepared and employed in the emulsion polymerization of vinyl acetate. The kinetic results reveal that the polymerization nucleation mechanism changes from homogeneous to micellar and RAFT-generated radicals can change the kinetic behavior from conventional emulsion polymerization to living radical polymerization. At higher concentrations of the modified RAFT agent, as a result of an aqueous phase reaction between RAFT and sulfate radicals, relatively more hydrophobic radicals are generated, which favors entry and propagation into micelles swollen with monomer. This observation was determined from the investigation of the polymerization rate and measurements of the average particle diameter and the number of particles per liter of the aqueous phase. Molecular weight analysis also demonstrated the participation of the RAFT agent in the polymerization in such a way as to restrict chain transfer reactions. This was determined by examining the evolution of polymer chain length and attaining higher molecular weights, even up to 50 % greater than the samples obtained from the conventional emulsion polymerization of vinyl acetate in the absence of the synthesized modified RAFT agent.

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington’s disease

BackgroundDespite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington’s disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the length-dependent nucleation and the nucleation-dependent elongation. In contrast to the repeat-length dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process.ResultsUsing polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression.ConclusionsOur findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.

Curcumin-glucoside, A Novel Synthetic Derivative of Curcumin, Inhibits α-Synuclein Oligomer Formation: Relevance to Parkinson's Disease

α-Synuclein aggregation is centrally implicated in Parkinson's disease (PD). It involves multi-step nucleated polymerization process via the formation of dimers, soluble toxic oligomers and insoluble fibrils. In the present study, we synthesized a novel compound viz., Curcumin-glucoside (Curc-gluc), a modified form of curcumin and studied its anti-aggregating potential with α-synuclein. Under aggregating conditions in vitro, Curc-gluc prevents oligomer formation as well as inhibits fibril formation indicating favorable stoichiometry for inhibition. The binding efficacies of Curc-gluc to both α-synuclein monomeric and oligomeric forms were characterized by micro-calorimetry. It was observed that titration of Curc-gluc with α-synuclein monomer yielded very low heat values with low binding while, in case of oligomers, Curc-gluc showed significant binding. Addition of Curc-gluc inhibited aggregation in a dosedependent manner and enhanced α-synuclein solubility, which propose that Curc-gluc solubilizes the oligomeric form by disintegrating preformed fibrils and this is a novel observation. Overall, the data suggest that Curc-gluc binds to α-synuclein oligomeric form and prevents further fibrillization of α-synuclein; this might aid the development of disease modifying agents in preventing or treating PD.

Perturbing the Central Hydrophobic Cluster (CHC) of Aβ(10-35) by Incorporation of Fluorinated Phenylalanine Derivatives

One of the putative causes of Alzheimer's disease involves aggregation of misfolded amyloid β (Aβ), a 39-42 residue polypeptide chain, and its subsequent deposition as amyloid plaques. The aggregation process proceeds via a nucleated polymerization mechanism where disordered peptide monomers interact with each other through hydrophobic interactions and rapidly extend and aggregate to eventually form larger fibrils with a highly ordered cross-strand β-sheet structure. It has also been suggested that the aromatic amino acid residues, tyrosine Y10 and phenylalanines (F19 and F20) in the central hydrophobic cluster (CHC) of the peptide play an important role in fibril assembly. In particular, F19 and F20 are suspected to be the drivers of the aggregation mechanism because of their hydrophobicity and aromaticity. In this context perturbation of the CHC through the introduction of non-natural (fluorinated) amino acids is expected to affect the aggregation process. Fluorinated amino acids in particular demonstrate distinct properties dictated by the presence of highly electronegative and hydrophobic fluorine atoms. However such fluorination is known to potentially eliminate the favorable interaction of aromatic hydrogens with the π-electron cloud, which can affect protein-protein interactions. In the present study the introduction of a pentafluoro-Phe in the hydrophobic core of the 26 residue Aβ peptide (Aβ10-35) and its effect on fibril formation has been investigated using circular dichroism (CD) and fluorescence methods which indicate a sequential conformational transition of the peptide from random coil → antiparallel β-sheets → parallel β-sheets. Transition time points have been obtained from these methods and compared to those obtained for the non-fluorinated peptide. UV resonance Raman (UVRR) studies have been performed to probe and characterize the vibrational modes of the fluoro-phenylalanines in the peptide and to explore their effect on the Phe-Phe π-stacking interactions.

Alpha-Synuclein Amyloid Oligomers Exhibit Beta-Sheet Antiparallel Structure as Revealed by FTIR Spectroscopy

Parkinson's disease is an aged-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-aa protein α-synuclein (AS). AS amyloid fibrils consist of interwound protofilaments with a cross β-sheet conformation. Fibrillation of AS follows a nucleation polymerization pathway involving major structural rearrangements and the population of diverse transient prefibrillar species varying in size and morphology. It is currently believed that cytotoxicity relies on these prefibrillar species rather than on the insoluble aggregates, although the underlying molecular mechanism remains elusive. Structural studies may contribute to the understanding of both amyloid aggregation mechanism and oligomer-induced toxicity.It is already recognized that soluble oligomeric AS species contain β-sheet structural elements that differ from the typical fibrillar structure. However, more detailed information on the origin of this difference is still lacking. In the present work we used ATR-FTIR spectroscopy, a technique especially sensitive to β-sheet structure, to discriminate between the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, as opposed to the parallel arrangement present in fibrils. We discussed about the possible regions that may be involved in such early β-sheet interactions.We suggest that antiparallel β-sheet structure might represent a distinctive signature of amyloid oligomers (Cerf et al, Biochem J, 2009, 421:415-23) underlying their common pathogenic action.

Nucleation of Sickle Hemoglobin Mixed with Hemoglobin A: Experimental and Theoretical Studies of Hybrid-Forming Mixtures

Sickle hemoglobin (HbS) is a point mutation of the two β subunits in normal Hb (HbA) that leads to nucleated polymerization and accompanying pathology. We measured the rates of homogeneous and heterogeneous nucleation of HbS in the presence of up to 50% HbA under conditions in which hybrid HbAS molecules will also form. The replacement of 50% of HbS by HbA slows polymerization by factors of ∼100 in the physiological range, which is substantially less than previously thought. To provide a theoretical description of these data, we extended the double nucleation model for HbS polymerization to conditions in which hybridized mixtures are present. Measurements of homogeneous nucleation and the theory agree only when at least one of the molecules in the nucleus is not a hybrid. We attribute this to the necessary presence in the nucleus of a molecule that utilizes both β-subunit mutation sites in intermolecular contacts, whereas the remaining molecules engage only one of the mutation sites. Heterogeneous nucleation appears to require an even greater number of nonhybrid molecules, presumably because of the need for the nucleus to attach to the polymer as well as to form internal bonds. These results also provide insights into the pathophysiology of sickle cell disease, including the occasional severe events that strike persons in whom both HbS and HbA are expressed, a condition known as sickle trait. The studies reported here are necessary for understanding physiologically relevant polymerization in the presence of ligands as well as therapeutically relevant copolymerizing inhibitors.

Physico-chemical and mechanical properties of nanocomposites prepared using cellulose nanowhiskers and poly(lactic acid)

A range of nanocomposites were prepared using cellulose nanowhiskers (CNWs) and poly(lactic acid) (PLA) via a solvent casting process. Acid hydrolysis process was used to produce CNWs from bleached cotton. Structural morphology and surface topography of the CNWs and nanocomposites were examined using transmission (TEM) and scanning electron microscopy. TEM images revealed rod-like whiskers in the nano-scale region which were dispersed within the PLA matrix. The presence of the functional groups of CNWs and PLA were confirmed via FTIR analysis. Tensile tests were conducted on thin films and the nanocomposites containing 1 wt% CNWs showed a 34 and 31% increase in tensile strength and modulus, respectively, compared to pure PLA. The dynamic mechanical analysis showed that the tensile storage modulus also increased in the visco-elastic temperature region with increasing CNWs content in the nanocomposites. Thermogravimetric analysis showed that all the materials investigated were thermally stable from room temperature to 210 °C. A positive effect of CNWs on the crystal nucleation of PLA polymer in the nanocomposites was observed using differential scanning calorimetry and X-ray diffraction analysis. The degradation profiles of the nanocomposites in deionised water over 1 week revealed a mass loss of 1.5–5.6% at alternate temperatures (25, 37 and 50 °C) and at the same conditions the swelling ratio and water uptake were seen to increase with CNWs content in the nanocomposites, which was strongly influenced by the presence of crystalline CNWs.

Nucleated Polymerisation in the Presence of Pre-Formed Seed Filaments

We revisit the classical problem of nucleated polymerisation and derive a range of exact results describing polymerisation in systems intermediate between the well-known limiting cases of a reaction starting from purely soluble material and for a reaction where no new growth nuclei are formed.

CS16-2. The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

Inflammasomes are caspase-1 activating, molecular inflammatory machines that proteolytically mature pro-inflammatory cytokines and induce pyroptotic cell death during innate immune responses. Recent structural studies of proteins that constitute inflammasomes have yielded fresh insights into their assembly mechanisms. In particular, these include a crystal structure of the CARD-containing NOD-like receptor NLRC4, the crystallographic and electron microscopy (EM) studies of the dsDNA sensors AIM2 and IFI16, and of the regulatory protein p202, and the cryo-EM filament structure of the PYD domain of the inflammasome adapter ASC. These data suggest inflammasome assembly that starts with ligand recognition and release of autoinhibition followed by step-wise rounds of nucleated polymerization from the sensors to the adapters, then to caspase-1. In this elegant manner, inflammasomes form by an ‘all-or-none’ cooperative mechanism, thereby amplifying the activation of caspase-1. The dense network of filamentous structures predicted by this model has been observed in cells as micron-sized puncta.

Nucleated polymerization with secondary pathways. III. Equilibrium behavior and oligomer populations

We explore the long-time behavior and equilibrium properties of a system of linear filaments growing through nucleated polymerisation. We show that the length distribution for breakable filaments evolves through two well defined limiting cases: first, a steady state distribution determined by the balance of breakage and elongation is reached; upon monomer depletion at the end of the growth phase, an equilibrium length distribution biased towards smaller filament fragments emerges. We furthermore compute the time evolution of the concentration of small oligomeric filament fragments. For frangible filaments, oligomers are present both at early times and at equilibrium, whereas in the absence of fragmentation, oligomers are only present in significant quantities at the beginning of the polymerisation reaction. Finally, we discuss the significance of these results for the biological consequences of filamentous protein aggregation.

Nucleated polymerization with secondary pathways. I. Time evolution of the principal moments

Self-assembly processes resulting in linear structures are often observed in molecular biology, and include the formation of functional filaments such as actin and tubulin, as well as generally dysfunctional ones such as amyloid aggregates. Although the basic kinetic equations describing these phenomena are well-established, it has proved to be challenging, due to their non-linear nature, to derive solutions to these equations except for special cases. The availability of general analytical solutions provides a route for determining the rates of molecular level processes from the analysis of macroscopic experimental measurements of the growth kinetics, in addition to the phenomenological parameters, such as lag times and maximal growth rates that are already obtainable from standard fitting procedures. We describe here an analytical approach based on fixed-point analysis, which provides self-consistent solutions for the growth of filamentous structures that can, in addition to elongation, undergo internal fracturing and monomer-dependent nucleation as mechanisms for generating new free ends acting as growth sites. Our results generalise the analytical expression for sigmoidal growth kinetics from the Oosawa theory for nucleated polymerisation to the case of fragmenting filaments. We determine the corresponding growth laws in closed form and derive from first principles a number of relationships which have been empirically established for the kinetics of the self-assembly of amyloid fibrils.

Nucleated polymerization with secondary pathways. II. Determination of self-consistent solutions to growth processes described by non-linear master equations

Nucleated polymerisation processes are involved in many growth phenomena in nature, including the formation of cytoskeletal filaments and the assembly of sickle hemoglobin and amyloid fibrils. Closed form rate equations have, however, been challenging to derive for these growth phenomena in cases where secondary nucleation processes are active, a difficulty exemplified by the highly non-linear nature of the equation systems that describe monomer dependent secondary nucleation pathways. We explore here the use of fixed point analysis to provide self-consistent solutions to such growth problems. We present iterative solutions and discuss their convergence behaviour. We establish a range of closed form results for linear growth processes, including the scaling behaviours of the maximum growth rate and of the reaction end-point. We further show that a self-consistent approach applied to the master equation of filamentous growth allows the determination of the evolution of the shape of the length distribution including the mean, the standard deviation, and the mode. Our results highlight the power of fixed-point approaches in finding closed form self-consistent solutions to growth problems characterised by the highly non-linear master equations.

Amyloid-β forms fibrils by nucleated conformational conversion of oligomers

Aβ amyloidogenesis is reported to occur via a nucleated polymerization mechanism, if so the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early non-fibrillar Aβ oligomers without observing amyloid fibrils, suggesting a mechanistic revision may be needed. Herein, we introduce Cys-Cys-Aβ1-40 that cannot bind to the latent fluorophore FlAsH as a monomer, but is capable of binding FlAsH as an non-fibrillar oligomer or as a fibril, rendering the conjugates fluorescent. FlAsH monitoring of Cys-Cys-Aβ1-40 aggregation provides compelling evidence that Aβ1-40 very rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism (seedable). Moreover, this methodology demonstrated that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of Aβ amyloidogenesis, while allowing fibril formation, rationalizing how low plasmalogen ethanolamine levels in the brain are epidemiologically linked to Alzheimer’s disease.

Benzene under High Pressure: a Story of Molecular Crystals Transforming to Saturated Networks, with a Possible Intermediate Metallic Phase

In a theoretical study, benzene is compressed up to 300 GPa. The transformations found between molecular phases generally match the experimental findings in the moderate pressure regime (<20 GPa): phase I (Pbca) is found to be stable up to 4 GPa, while phase II (P4(3)2(1)2) is preferred in a narrow pressure range of 4-7 GPa. Phase III (P2(1)/c) is at lowest enthalpy at higher pressures. Above 50 GPa, phase V (P2(1) at 0 GPa; P2(1)/c at high pressure) comes into play, slightly more stable than phase III in the range of 50-80 GP, but unstable to rearrangement to a saturated, four-coordinate (at C), one-dimensional polymer. Actually, throughout the entire pressure range, crystals of graphane possess lower enthalpy than molecular benzene structures; a simple thermochemical argument is given for why this is so. In several of the benzene phases there nevertheless are substantial barriers to rearranging the molecules to a saturated polymer, especially at low temperatures. Even at room temperature these barriers should allow one to study the effect of pressure on the metastable molecular phases. Molecular phase III (P2(1)/c) is one such; it remains metastable to higher pressures up to ∼200 GPa, at which point it too rearranges spontaneously to a saturated, tetracoordinate CH polymer. At 300 K the isomerization transition occurs at a lower pressure. Nevertheless, there may be a narrow region of pressure, between P = 180 and 200 GPa, where one could find a metallic, molecular benzene state. We explore several lower dimensional models for such a metallic benzene. We also probe the possible first steps in a localized, nucleated benzene polymerization by studying the dimerization of benzene molecules. Several new (C(6)H(6))(2) dimers are predicted.

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization

Chromatin domains are believed to spread via a polymerization-like mechanism in which modification of a given nucleosome recruits a modifying complex, which can then modify the next nucleosome in the polymer. In this study, we carry out genome-wide mapping of the Sir3 component of the Sir silencing complex in budding yeast during a time course of establishment of heterochromatin. Sir3 localization patterns do not support a straightforward model for nucleation and polymerization, instead showing strong but spatially delimited binding to silencers, and weaker and more variable Ume6-dependent binding to novel secondary recruitment sites at the seripauperin (PAU) genes. Genome-wide nucleosome mapping revealed that Sir binding to subtelomeric regions was associated with overpackaging of subtelomeric promoters. Sir3 also bound to a surprising number of euchromatic sites, largely at genes expressed at high levels, and was dynamically recruited to GAL genes upon galactose induction. Together, our results indicate that heterochromatin complex localization cannot simply be explained by nucleation and linear polymerization, and show that heterochromatin complexes associate with highly expressed euchromatic genes in many different organisms.