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Amyloid β (Aβ) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aβ1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1–5 of Aβ with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aβ1-42 oligomers and higher aggregates such as fibrils but discriminate against Aβ1-42 monomer species. We find that for detection of Aβ1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aβ) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aβ variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects.

A novel surface modification technique for grafting alkyl methacrylate monomers from commercial poly(ether sulfone) (PES) nanofiltration membranes is developed through a combination of helium and oxygen atmospheric pressure plasma treatment followed by Activators Regenerated by Electron Transfer (ARGET) Atom Transfer Radical Polymerization (ATRP). The resulting membrane surfaces show degree of grafting increases of 28%, 94%, and 270% for methyl methacrylate (C1), hexyl methacrylate (C6), and stearyl methacrylate (C18), respectively, when characterized with Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy. Scanning Electron Microscopy and Atomic Force Microscopy (AFM) show a rippled, fibrous morphology for the PES membranes grafted with C18 and reinforced through molecular dynamics simulations. AFM of the PES membranes grafted with C18 show an increase of ~ 23% in root-mean square (RMS) roughness as well as 4x higher adhesion force when probed with a hydrophobic gold cantilever tip when compared with the unmodified PES membranes, confirming a successful surface grafting reaction and increase in surface hydrophobicity, respectively. This technique allows enhanced synthesis of polymer grafted membranes using relatively green reaction solvent and enables “structure-by-design” surface morphology control with future applications in membrane separation processes such as organic solvent nanofiltration, gas separations, and desalination.

This work addresses the important link between the microstructure of a membrane and its filtration performance. 2D computational fluid and particle drag mechanics are combined with particle and membrane force measurements in aqueous solutions containing inorganic ions to study particle intrusion and capture in microporous commercial polymer and computer-generated teardrop membranes. Fits of the DLVO theory to force-distance profiles obtained membrane surface potentials needed for the computations. In silico predictions of particle intrusion for a commercial membrane qualitatively agree with experimental filtration measurements using scanning electron microscopy with particle tracking via energy dispersive X-ray spectroscopy. Highlighting the poor flow field, several dominant inhomogeneous 2D flow conduits with large unused regions of the internal pore structure are discovered. To guide improved design, new computer-generated microporous teardrop structures that can equalize the flow field, adjust the tortuosity of the flow path and vary the reactivity of the surface were tested in silico. The main assumptions of the computational model were that 2D flows are a valid description of 3D flows, all forces were applied at the particle center of mass, forces were calculated based on the physical diameter of the spherical particles. Relatively large pores (~5 μm) and large particles (~1 μm) were selected for easy detection and analysis. However, the computational fluid and particle flow analysis and the inter-surface forces scale independently with size and should apply at all classical dimensions (i.e. for nano, ultra and microfiltration). Assumptions for the intermolecular force measurements were that electrostatic and van der Waal's forces dominated and hence that the DLVO theory was valid and that the zeta potential values were close to those at the wall (i.e. surface potential). In particular the DLVO was applied to ideal geometries: a sphere (i.e. AFM probe) near to a flat surface (i.e. either a silica wafer or a hot pressed PES membrane). To our knowledge, this is the first attempt combining particle drag mechanics with intermolecular force measurements to help explain particle dynamics in synthetic membranes. This computational fluid mechanics-based tool can be used to characterize membranes for separation performance and guide improved design, synthesis and testing of new microporous membranes.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

Protein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest.In vitromethods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

The objective of this work is the development of a process for the purification of lectins with affinity membranes. To this aim affinity membranes were prepared by chemical modification of a cellulose matrix. Different ligands were tested endowed with the different affinity towards the lectins used. As a model protein a lectin obtained by chromatographic techniques from Momordica charantia seeds was mainly used; Peanut agglutinin and Ricinus communis agglutinin were also considered. Among the various ligands tested N -acetyl- d -galactosamine gave the best separation performances, whilst arabinogalactan gave the highest binding capacity. The ligand immobilized on the membrane surface is quantified indirectly by measuring the amount of protein bound to the membrane. The kinetics of adsorption and desorption of the purification process has been studied in detail for the different supports. Modified membranes have been used in separation process of lectins with good results in terms of binding capacity towards the protein of interest.

The role and mechanism of amyloid proteins in neurodegenerative diseases remains an unsolved problem. Recent findings have shown that low-molecular-weight oligomers, generated during the early stage of the nucleation process, are the most neurotoxic species – for example, Aβ peptide oligomers for Alzheimer’s disease. To address this challenge, researchers have attempted to isolate, identify, quantify and characterize these small oligomeric species using a series of characterization and high-resolution separation methods. Here we present an overview of the progress reported during the past decade regarding oligomers of a model amyloid protein, insulin. Although the behavior and properties of these oligomers during conversion from the folded insulin monomer through oligomers to fibers is not widely accepted, several conclusions are possible. Insulin proceeds to aggregate in vitro under harsh conditions (e.g. pH 1.6, 65°C) along a universal path independent of kinetics, from monomers to dimers to larger entities, such as nuclei comprising hexamers, and then to fibrils. Amyloidogenic oligomers occur in very low concentrations and, in some cases, at undetectable levels. Their aggregation is strongly influenced by different initial conditions. None of the techniques reviewed here was able to provide a detailed and complete description of the oligomeric species present because each technique suffered from intrinsic limitations. In summary, isolation and purification of these ephemeral oligomer species has proved to be very difficult due to their low number and strong inherent tendency to aggregate under increased shear, ionic strength, temperature and hydrogen ion concentration.

Amyloid diseases, including Alzheimer's and Parkinson's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, beta-sheet rich fibrils. There are many theories on the species that causes toxicity in amyloid diseases, mainly focused on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, which makes their full characterization difficult. We have isolated a toxic protein species that acts like an oligomer and may provide evidence of a stable oligomer. This oligomer was isolated by dissolving amyloid fibrils at high pH, then purified and concentrated by diafiltration. It has a mass greater than 100 kDa and a diameter ranging of 48 ± 15 nm. The oligomer seeds the formation of fibrils in a dose dependent manner and exhibits Thioflavin-T fluorescence, but circular dichroism and deep UV resonance Raman spectroscopy did not find any evidence of an increase in beta-sheet structure. It appears that this oligomer is largely unstructured protein. We hypothesize that the oligomer does not decompose at high pH and maintains its structure in solution. All of the insulin, however, that joined the oligomer to elongate the beta-sheet rich fibrils folded in a different conformation and could be removed from the fibril and returned to native, dissolved insulin. This is the first time that a stable oligomer of an amyloid reaction has been separated and characterized without genetically engineering the protein or having additives in the fibrillation media. It appears that the oligomer does not have the same structure as the fibrils and is possibly intrinsically unfolded. This may make the search for a stable amyloid oligomer or nucleus even more difficult since methods to detect beta-sheets are often employed in the search for these structures.

Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimer's and Parkinson's. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre-treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small-angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre-treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem.

Maghemite (γ-Fe2O3) magnetic nanoparticles of 15.0 ± 2.1 nm were formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Human insulin amyloid fibrils were formed by incubating the monomeric insulin dissolved in aqueous continuous phase at pH 1.6 and 65°C. Magnetic human insulin amyloid fibrils/γ-Fe2O3 nanoparticle assemblies were prepared by interacting the γ-Fe2O3 nanoparticles with the insulin amyloid fibrils during or after their formation. The nanoparticles attached selectively to the insulin fibrils in both cases. The kinetics of the insulin fibrillation process in the absence and the presence of the γ-Fe2O3 nanoparticles was elucidated. The insulin amyloid fibrils/γ-Fe2O3 nanoparticle assemblies were easily extracted from the aqueous phase via a magnetic field. We hypothesize that this selective extraction approach may also be applicable for the removal of other amyloidogenic proteins that lead to neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington's, mad cow, and prion diseases) from their continuous phase, e.g. milk, blood, neurological fluid, etc. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

To help identify the etiological agents for amyloid-related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H2O- and D2O-based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end-on-end association of dimers to form three-dimer nuclei, and then exhibits broadening or side-on-side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics. Proteins 2009. © 2008 Wiley-Liss, Inc.

Abeta binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Abeta is likely a direct link between Abeta and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Abeta interaction. A van't Hoff analysis revealed that the ABAD-Abeta association is driven by a favorable entropic change (DeltaS = 300 +/- 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (DeltaH = 49 +/- 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Abeta interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Abeta. SPR confirmed the conformational changes in the ABAD-Abeta complex after Abeta binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Abeta complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Abeta on the ABAD-NAD interaction. Conversely, NAD inhibits the Abeta-ABAD interaction. Binding of Abeta and binding of NAD to ABAD are likely mutually exclusive. Thus, Abeta binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Abeta toxicity in Alzheimer's disease.

The specific interaction between MBP-fusion proteins and amylose based affinity membranes has been presented recently. In this work, the attention is focused on the influence of ionic strength on the equilibrium isotherm and on the kinetics of adsorption and desorption. Three different MBP-fusion proteins have been used: MBP-rubredoxin (MW 51 kDa), MBP-intein-CBD (MW 97 kDa) and MBP-β galactosidase (MW 160 kDa) characterized by different dimensions. The equilibrium data follow the Langmuir isotherm, whose parameters qm and Kd show a dependence on salt concentration in the feed solution. By increasing the ionic strength, the binding capacity at saturation qm decreases while the equilibrium constant Kd increases, indicating that a higher concentration of NaCl reduces the affinity interaction between protein and ligand. A simple kinetic model has been investigated for the interpolation of adsorption and elution curves. No mass transfer effects have been considered within the liquid phase and inside the pores of the stationary phase. The high processing speed of membrane affinity filtration has been exploited. The kinetics of adsorption and elution have been described and the values of the kinetic constants have been obtained both for adsorption and elution conditions for all proteins. The influence of the action of a competing substrate like maltose on the removal of the immobilized product has been also determined.

BACKGROUND: In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. METHODOLOGY/PRINCIPAL FINDINGS: Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei CONCLUSIONS: Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases.

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

The molecular rearrangement of soluble proteins into fibers is a common attribute of amyloid diseases: Alzheimer's disease, Parkinson's disease, spongiform encephalopathies (including mad cow disease), and other prion diseases. In the past century considerable progress has been made in characterizing amyloid diseases, but the connection between amyloidosis and the disease is still unclear: Contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. The debate is still open.Here, we offer the first attempt to “reverse-estimate” the concentrations of nuclei, starting from a distribution of fibril lengths. Assuming the nucleation model is valid, with a few reasonable assumptions, a fibril length distribution and a set of seeding experiments, we estimated the in vitro concentration of nuclei for the model hormone, recombinant human insulin, to be in the picomolar range. Fibril lengths are measured with an atomic force microscope and seeding shows that fibrils of different lengths exhibit similar growth rates. Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in fractionating, isolating and blocking nuclei toxicity. Moreover, this theoretical approach, based on our measurements and a structural fibril model recently published by David Eisenberg's group at UCLA, is general and could be used for other amyloid proteins.