amyloid-beta-peptides and hexafluoroisopropanol

Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ1-40 and 1.6±0.1 nm for Aβ1-42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1-40 and 3.2±0.4 nm for Aβ1-42 including a surface layer of dHFIP solvent molecules.

Topics: Amyloid beta-Peptides; Humans; Hydrodynamics; Hydrogen Bonding; Models, Chemical; Models, Molecular; Neutron Diffraction; Peptide Fragments; Propanols; Protein Aggregation, Pathological; Protein Conformation; Scattering, Small Angle; Solvents

Fluorinated alcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have the ability to promote α-helix and β-hairpin structure in proteins and peptides. HFIP has been used extensively to dissolve various amyloidogenic proteins and peptides including Aβ, in order to ensure their monomeric status. In this paper, we have investigated the self-assembly of Aβ40, Aβ42, and Aβ43 in aqueous mixtures of fluorinated alcohols from freshly dissolved stock solutions in HFIP. We have observed that formation of fibrillar and non-fibrillar structures are dependent on the solvent composition. Peptides form fibrils with ease when reconstituted in deionized water from freshly dissolved HFIP stocks. In aqueous mixtures of fluorinated alcohols, either predominant fibrillar structures or clustered aggregates were observed. Aqueous mixtures of 20% HFIP are more favourable for Aβ fibril formation as compared to 20% TFE. When Aβ40, Aβ42, and Aβ43 stocks in HFIP are diluted in 50% aqueous mixtures in phosphate buffer or deionized water followed by slow evaporation of HFIP, Aβ peptides form fibrils in phosphate buffer and deionized water. The clustered structures could be off-pathway aggregates. Aβ40, Aβ42, and Aβ43 showed significant α-helical content in freshly dissolved HFIP stocks. The α-helical conformational intermediate in Aβ40, Aβ42, and Aβ43 could favour the formation of both fibrillar and non-fibrillar aggregates depending on solvent conditions and rate of α-helical to β-sheet transition.

Topics: Amyloid beta-Peptides; Circular Dichroism; Halogenation; Humans; Microscopy, Electron, Transmission; Peptide Fragments; Propanols; Protein Conformation; Solvents; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Trifluoroethanol

Alzheimer's disease is characterized by the abnormal aggregation of amyloid-β (Aβ)1-40 and Aβ1-42 peptides into fibrils. In this work, we analyzed the kinetics of Aβ1-40 and Aβ1-42 fibril formation in vitro using Thioflavin T fluorescence. We synthesized high-purity peptides and performed a hexafluoro-2-propanol pre-treatment to yield uniform peptide solutions as starting materials. We found that the aggregation is clearly affected by the presence of sub-millimolar quantities of antibodies against the C-terminal region of the peptides. Because the fibrillization of these peptides is closely related to the pathogenesis of Alzheimer's disease, blocking this process may provide significant therapeutic benefit.

Topics: Amyloid; Amyloid beta-Peptides; Antibodies; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Microscopy, Electron, Transmission; Peptide Fragments; Propanols; Time Factors

Deposition of insoluble fibrillar aggregates of β-amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α-helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α-helical to β-conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring-like structures. Aβ16-22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α-helical conformation in Aβ16-22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16-22 are composed of the peptides in β-conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid-like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16-22 in HFIP results in the formation of annular amyloid-like structures.

Topics: Amyloid beta-Peptides; Benzothiazoles; Circular Dichroism; Fluorescent Dyes; Humans; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Nanostructures; Particle Size; Peptide Fragments; Propanols; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared; Thiazoles

Understanding the pathogenicity of amyloid-beta (Abeta) peptides constitutes a major goal in research on Alzheimer's disease (AD). One hypothesis entails that Abeta peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Abeta-induced ion flux in AD. The work presented here addresses two prevalent controversies regarding the nature of transmembrane ion flux induced by Alphabeta peptides. First, the results clarify that Alphabeta can induce stepwise ion flux across planar lipid bilayers as opposed to a gradual increase in transmembrane current; they show that the previously reported gradual thinning of membranes with concomitant increase in transmembrane current arises from residues of the solvent hexafluoroisopropanol, which is commonly used for the preparation of amyloid samples. Second, the results provide additional evidence suggesting that Abeta peptides can induce ion channel-like ion flux in cellular membranes that is independent from the postulated ability of Alphabeta to modulate intrinsic cellular ion channels or transporter proteins.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Biophysics; Cell Membrane; Cells, Cultured; Cerebral Cortex; Embryo, Mammalian; Female; Humans; Ion Channel Gating; Ion Transport; Ions; Lipid Bilayers; Male; Membrane Potentials; Membranes, Artificial; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neuroblastoma; Neurons; Patch-Clamp Techniques; Peptide Fragments; Presenilin-1; Propanols

The structure and aggregation state of amyloid beta-peptide (Abeta) in membrane-like environments are important determinants of pathological events in Alzheimer's disease. In fact, the neurotoxic nature of amyloid-forming peptides and proteins is associated with specific conformational transitions proximal to the membrane. Under certain conditions, the Abeta peptide undergoes a conformational change that brings the peptide in solution to a "competent state" for aggregation. Conversion can be obtained at medium pH (5.0-6.0), and in vivo this appears to take place in the endocytic pathway. The combined use of (1)H NMR spectroscopy and molecular dynamics-simulated annealing calculations in aqueous hexafluoroisopropanol simulating the membrane environment, at different pH conditions, enabled us to get some insights into the aggregation process of Abeta, confirming our previous hypotheses of a relationship between conformational flexibility and aggregation propensity. The conformational space of the peptide was explored by means of an innovative use of principal component analysis as applied to residue-by-residue root-mean-square deviations values from a reference structure. This procedure allowed us to identify the aggregation-prone regions of the peptide.

Topics: Amyloid beta-Peptides; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Peptide Fragments; Principal Component Analysis; Propanols; Protein Conformation; Solutions

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.

Topics: Amyloid beta-Peptides; Circular Dichroism; Humans; Micelles; Models, Molecular; Peptide Fragments; Propanols; Protein Structure, Secondary; Sodium Dodecyl Sulfate

Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered structures that appear to assemble according to ordered mechanisms. Some of these fibrils, as illustrated here with the Alzheimer's plaque peptide amyloid beta, assemble to an endpoint that is a dynamic equilibrium between monomers and fibrils exhibiting a characteristic equilibrium constant with an associated free energy of formation. Some fibrils, as illustrated here with the polyglutamine repeat sequences associated with Huntington's disease, assemble via highly regular mechanisms exhibiting nucleated growth polymerization kinetics. Here, we describe a series of linked methods for quantitative analysis of such aggregation kinetics and thermodynamics, focusing on a robust high-performance liquid chromatography (HPLC)-based sedimentation assay. An integrated group of protocols is provided for peptide disaggregation, setting up the HPLC sedimentation assay, the preparation of fibril seed stocks and determination of the average functional molecular weight of the fibrils, elongation and nucleation kinetics analysis, and the determination of the critical concentration describing the thermodynamic endpoint of fibril elongation.

Topics: Amyloid; Amyloid beta-Peptides; Chromatography, High Pressure Liquid; Kinetics; Peptide Chain Elongation, Translational; Peptide Fragments; Peptides; Propanols; Protein Structure, Quaternary; Temperature; Thermodynamics; Trifluoroacetic Acid

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Benzothiazoles; Biochemistry; Chromatography; Circular Dichroism; Humans; Ions; Kinetics; Light; Microscopy, Atomic Force; Microscopy, Electron; Peptide Fragments; Propanols; Protein Conformation; Scattering, Radiation; Thiazoles; Time Factors

The misfolding of the amyloid peptide, which is the result of a well-known alpha-to-beta transition, causes neurodegenerative disorder. Fluorinated alcohols have been described in the literature as potent solvents which can refold the beta-conformation. The present studies demonstrate the effectiveness of differently fluorinated alcohols for the beta-to-alpha refolding process on fibrillar aggregated amyloid beta(1-40). The regenerated helical structure is shown to be maintained in the absence of the fluoroalcohols, a behaviour which was found to contrast with immunoglobulin. We interpret this difference on the basis of the hydrophilic/hydrophobic domains in the amyloid sequence and present some speculations regarding the free-energy levels of the folded states of both proteins. The effect of the -CF(3) group on the observed conformational changes is interpreted as a result of alterations of the hydration shell of the peptides. Moreover, based on the results achieved with fluoroalcohols, we have used novel fluorinated amphiphiles possessing blood-compatibility properties and studied their effect on amyloid beta(1-40). First results point in the direction of a beta-to-alpha transition. Therefore, the use of fluorine groups in the development of new drugs is considered a new possibility requiring further investigation for the prevention of amyloidosis.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Amyloidosis; Circular Dichroism; Drug Design; Ethanol; Humans; Hydrophobic and Hydrophilic Interactions; Microscopy, Atomic Force; Molecular Sequence Data; Peptide Fragments; Propanols; Protein Conformation; Protein Folding; Protein Structure, Secondary; Solutions; Trifluoroethanol