Inhibition of amyloid fibril formation of human amylin by N-alkylated amino acid and alpha-hydroxy acid residue containing peptides

Amyloid deposits are formed as a result of uncontrolled aggregation of (poly)peptides or proteins. Today several diseases are known, for example Alzheimer's disease, Creutzfeldt-Jakob disease, mad cow disease, in which amyloid formation is involved. Amyloid fibrils are large aggregates of beta-pleated sheets and here a general method is described to introduce molecular mutations in order to achieve disruption of beta-sheet formation. Eight backbone-modified amylin derivatives, an amyloidogenic peptide involved in maturity onset diabetes, were synthesized. Their beta-sheet forming properties were studied by IR spectroscopy and electron microscopy. Modification of a crucial amide NH by an alkyl chain led to a complete loss of the beta-sheet forming capacity of amylin. The resulting molecular mutated amylin derivative could be used to break the beta-sheet thus retarding beta-sheet formation of unmodified amylin. Moreover, it was found that the replacement of this amide bond by an ester moiety suppressed fibrillogenesis significantly. Introduction of N-alkylated amino acids and/or ester functionalities-leading to depsipeptides-into amyloidogenic peptides opens new avenues towards novel peptidic beta-sheet breakers for inhibition of beta-amyloid aggregation.     

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.     

Unraveling the mechanism of nanotube formation by chiral self-assembly of amphiphiles

The self-assembly of nanotubes from chiral amphiphiles and peptide mimics is still poorly understood. Here, we present the first complete path to nanotubes by chiral self-assembly studied with C(12)-β(12) (N-α-lauryl-lysyl-aminolauryl-lysyl-amide), a molecule designed to have unique hybrid architecture. Using the technique of direct-imaging cryo-transmission electron microscopy (cryo-TEM), we show the time-evolution from micelles of C(12)-β(12) to closed nanotubes, passing through several types of one-dimensional (1-D) intermediates such as elongated fibrils, twisted ribbons, and coiled helical ribbons. Scattering and diffraction techniques confirm that the fundamental unit is a monolayer lamella of C(12)-β(12), with the hydrophobic tails in the gel state and β-sheet arrangement. The lamellae are held together by a combination of hydrophobic interactions, and two sets of hydrogen-bonding networks, supporting C(12)-β(12) monomers assembly into fibrils and associating fibrils into ribbons. We further show that neither the "growing width" model nor the "closing pitch" model accurately describe the process of nanotube formation, and both ribbon width and pitch grow with maturation. Additionally, our data exclusively indicate that twisted ribbons are the precursors for coiled ribbons, and the latter structures give rise to nanotubes, and we show chirality is a key requirement for nanotube formation.     

Tuning secondary structure and self-assembly of amphiphilic peptides

Self-assembly is one of nature's mechanisms by which higher order structures are obtained. Two of the main driving forces for self-assembly, hydrophobic interactions and hydrogen bonding, are both present within amphiphilic peptides. Here, it is demonstrated how the intricately interconnected folding and assembly behavior of an N-terminally acylated peptide, with the sequence GANPNAAG, has been tuned by varying its hydrophobic tail and thermal history. The change in interplay between hydrophobic forces and peptide folding allowed the occurrence of different types of aggregation, from soluble peptides with a random coil conformation to aggregated peptides arranged in a beta-sheet assembly, which form helically twisted bilayer ribbons.     

Phase networks of cross-β peptide assemblies

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimer's disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies.     

Interaction between synthetic amyloid-beta-peptide (1-40) and its aggregation inhibitors studied by electrospray ionization mass spectrometry.

It is generally postulated that amyloid-beta-peptides play a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathological properties of these peptides, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of amyloid-beta-peptides to form beta-sheet structures and/or amyloid fibrils. Amyloid-beta-peptides are known to aggregate spontaneously in vitro with the formation of amyloid fibrils. The intervention on the amyloid-beta-peptides aggregation process can be envisaged as an approach to stopping or slowing the progression of Alzheimer's disease. In the last few years a number of small molecules have been reported to interfere with the in vitro aggregation of amyloid-beta-peptides. Melatonin, a hormone recently found to protect neurons against amyloid-beta-peptide toxicity, interacts with amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and inhibits the progressive formation of beta-sheet and/or amyloid fibrils. These interactions between melatonin and the amyloid peptides have been demonstrated by circular dichroism (CD) and electron microscopy for amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and by nuclear magnetic resonance (NMR) spectroscopy for amyloid-beta-peptide (1-40). Our electrospray ionization mass spectrometric (ESI-MS) studies also proved that there is a hydrophobic interaction between amyloid-beta-peptide (1-40) and melatonin and the proteolytic investigations suggested that the interaction took place on the 29-40 amyloid-beta-peptide segment. The wide-ranging application of these results would provide further information and help in biological research.     

Morphological control of helical solid bilayers in high-axial-ratio nanostructures through binary self-assembly

Mixed molecular species of cardanyl glucoside derived from renewable resources provide nanotubes upon self-assembly in water, while the saturated homologue generated a twisted fibrous morphology. The cardanyl glucoside mixture was fractionated into four individual components in order to study their contribution to the nanotube formation. The rational control of self-assembled helical morphologies was achieved by binary self-assembling of the saturated and monoene derivatives. This method can generate a diversity of self-assembled high-axial-ratio nanostructures (HARNs), ranging from twisted ribbons and helical ribbons to nanotubes.     

Nanotube and three-way nanotube formation with nonionic amphiphilic block peptides

Amphiphilic block polypeptides having a helical hydrophobic block with a uniform chain length and a hydrophilic nonionic block were newly synthesized and self-assembled into homogeneous nanotubes with ca. 60 nm diameter and ca. 200 nm length. The tubular assembly was shown to be elongated by heating over micrometer length without changing the diameter. Notably, a distinctive three-way nanotube was obtained just by mixing two kinds of amphiphilic polypeptides with the same helical hydrophobic block but different chain lengths of the hydrophilic block. The morphology of the molecular assemblies was shown to be tunable from a curved sheet-shaped assembly to a long or short nanotubular assembly and a three-way nanotubular assembly by suitable molecular design of the hydrophobic block, selection of the chain length of the hydrophilic block, mixing two-type block peptides, and processing such as heating.     

Solvent-induced helix superstructure in achiral dumbbell-shaped hydrazine derivatives

We studied hydrogen-bonding assemblies in a series of dumbbell-shaped hydrazine derivatives, namely oxalyl N',N'-bis(3,4-dialkoxybenzoyl)-hydrazide (BFH-n, n = 4, 6, 8, 10) and oxalyl N',N'-dibenzoyl-hydrazide (FH-0). It has been demonstrated that NH-1 protons of BFH-n precipitated from tetrahydrofuran (THF) or dimethylformamide (DMF) were involved in intramolecular H-bonding to form 6-membered rings. Meanwhile, NH-2 protons of BFH-n precipitated from THF formed intermolecular hydrogen bonds with C═O groups of neighboring molecules, while NH-2 protons of BFH-n precipitated from DMF formed intermolecular hydrogen bonds with C═O group of neighboring DMF molecules. C═O, -CH(3), and -CH groups of DMF molecules participated in multiple intermolecular hydrogen bonds with the -N-H and -C═O groups of FH-0 molecules in single-crystals formed in DMF, leading to a double helix morphology with a pitch of 24.2 ? along the c direction. Both left- and right-handed helical micrometer-length ribbons with nonuniform helical pitches were observed in an achiral BFH-10 xerogel precipitated from DMF.     

Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity

Small amphiphilic peptides are attractive building blocks to design biocompatible supramolecular structures via self-assembly, with applications in, for example, drug delivery, tissue engineering, and nanotemplating. We address the influence of systematical changes in the amino acid sequence of such peptides on the self-assembled macromolecular structures. For cationic-head surfactant-like eight-residue peptides, the apolar tail amino acids were chosen to systematically vary the propensity to form an alpha-helical secondary structure while conserving the overall hydrophobicity of the sequence. Characterization of the supramolecular structures indicates that for short peptides a beta-sheet secondary structure correlates with ribbonlike assemblies while random-coil and alpha-helical secondary structures correlate with assembly of rods.     

Controlling the morphology of cross beta-sheet assemblies by rational design

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.     

Mirror image supramolecular helical tapes formed by the enantiomeric-depsipeptide derivatives of the amyloidogenic peptide amylin(20-29)

Factors that determine the chirality of supramolecular helical tapes formed by a backbone-modified amylin(20-29) depsipeptide and inverso-depsipeptide, were studied by Fourier transform infrared spectroscopy, circular dichroism and transmission electron microscopy. Although β-sheet propensity was absent in both peptides, it was found that the l-depsipeptide formed left-handed and the enantiomeric d-depsipeptide right-handed helical tapes. Moreover, the backbone-modified depsipeptides, showed a certain degree of cross-recognition between both enantiomers, which might have implications in designing amyloid formation inhibitors.     

Self-assembly of beta-sheet forming peptides into chiral fibrillar aggregates

The authors introduce a novel mid-resolution off-lattice coarse-grained model to investigate the self-assembly of beta-sheet forming peptides. The model retains most of the peptide backbone degrees of freedom as well as one interaction center describing the side chains. The peptide consists of a core of alternating hydrophobic and hydrophilic residues, capped by two oppositely charged residues. Nonbonded interactions are described by Lennard-Jones and Coulombic terms. The influence of different levels of "hydrophobic" and "steric" forces between the side chains of the peptides on the thermodynamics and kinetics of aggregation was investigated using Langevin dynamics. The model is simple enough to allow the simulation of systems consisting of hundreds of peptides, while remaining realistic enough to successfully lead to the formation of chiral, ordered beta tapes, ribbons, as well as higher order fibrillar aggregates.     

Amyloid formation from an α-helix peptide bundle is seeded by 3(10)-helix aggregates

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water‐soluble, α‐helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole‐containing macrocyclic scaffold. The resulting 4α‐helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration‐dependent conversion to β‐sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5–50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α‐helix bundles. During α→β folding we were able to detect soluble 3₁₀ helices in solution by using 2D‐NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3₁₀‐helix aggregation triggered in this case by template‐promoted helical bundling and by hydrogen‐bonding glutamic acid side chains. A mechanism involving α?α₄?(3₁₀)₄?(3₁₀)_n?(β)_n?m(β)_n equilibria is plausible for this peptide and also for peptides lacking hydrogen‐bonding side chains, with unfavourable equilibria slowing the α→β conversion.     

Estimation of electrokinetic and hydrodynamic global properties of relevant amyloid‐beta peptides through the modeling of their effective electrophoretic mobilities and analysis of their propensities to aggregation

Neuronal activity loss may be due to toxicity caused by amyloid‐beta peptides forming soluble oligomers. Here amyloid‐beta peptides (1–42, 1–40, 1–39, 1–38, and 1–37) are characterized through the modeling of their experimental effective electrophoretic mobilities determined by a capillary zone electrophoresis method as reported in the literature. The resulting electrokinetic and hydrodynamic global properties are used to evaluate amyloid‐beta peptide propensities to aggregation through pair particles interaction potentials and Brownian aggregation kinetic theories. Two background electrolytes are considered at 25°C, one for pH 9 and ionic strength I = 40 mM (aggregation is inhibited through NH₄OH) the other for pH 10 and I = 100 mM (without NH₄OH). Physical explanations of peptide oligomerization mechanisms are provided. The effect of hydration, electrostatic, and dispersion forces in the amyloidogenic process of amyloid‐beta peptides (1–40 and 1–42) are quantitatively presented. The interplay among effective charge number, hydration, and conformation of chains is described. It is shown that amyloid‐beta peptides (1–40 and 1–42) at pH 10, I = 100 mM and 25°C, may form soluble oligomers, mainly of order 2 and 4, after an incubation of 48 h, which at higher times evolve and end up in complex structures (protofibrils and fibrils) found in plaques associated with Alzheimer's disease.     

Helically chiral ferrocene peptides containing 1'-aminoferrocene-1-carboxylic acid subunits as turn inducers

We present a detailed structural study of peptide derivatives of 1'-aminoferrocene-1-carboxylic acid (ferrocene amino acid, Fca), one of the simplest organometallic amino acids. Fca was incorporated into di- to pentapeptides with D- and L-alanine residues attached to either the carboxy or amino group, or to both. Crystallographic and spectroscopic studies (circular dicroism (CD), IR, and NMR) of about two dozen compounds were used to gain a detailed insight into their structures in the solid state as well as in solution. Four derivatives were characterized by single-crystal X-ray analysis, namely Boc-Fca-Ala-OMe (16), Boc-Fca-D-Ala-OMe (17), Boc-Fca-beta-Ala-OMe (18), and Boc-Ala-Fca-Ala-Ala-OMe (21) (Boc=tert-butyloxycarbamyl). CD spectroscopy is an extremely useful tool to elucidate the helical chirality of the metallocene core. Unlike in all other known ferrocene peptides, the helical chirality of the ferrocene is governed solely by the chirality of the amino acid attached to the N terminus of Fca. Depending on the degree of substitution of both cyclopentadiene (Cp) rings, different hydrogen-bonding patterns are realized. (1)H NMR and IR spectroscopy, together with the results from X-ray crystallography, give detailed information regarding not only the hydrogen-bonding patterns of the compounds, but also the equilibria between different conformers in solution. Differences in chemical shifts of NH protons in dimethyl sulfoxide ([D(6)]DMSO) and CDCl(3), that is, the variation ratio (vr), is used for the first time as a measure of the hydrogen-bonding strength of individual COHN bonds in ferrocenoyl peptides. In dipeptides with one intramolecular hydrogen bond between the pendant chains, for example, in dipeptide 16, an equilibrium between hydrogen-bonded and open forms is observed, as testified by a vr value of around 0.5. Higher peptides, such as tetrapeptide 21, are able to form two intramolecular hydrogen bonds stabilizing one single conformation in CDCl(3) solution (vr approximately 0). Due to the low barrier of Cp-ring rotation, new and unnatural hydrogen-bonding patterns are emerging. The systematic work described herein lays a solid foundation for the rational design of metallocene peptides with unusual structures and properties.     

Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides

Fabrication of controlled peptide nanofibers with homogeneous morphology has been demonstrated. Amphiphilic beta-sheet peptides were designed as sequences of Pro-Lys-X(1)-Lys-X(2)-X(2)-Glu-X(1)-Glu-Pro. X(1) and X(2) were hydrophobic residues selected from Phe, Ile, Val, or Tyr. The peptide FI (X(1)=Phe; X(2)=Ile) self-assemble into straight fibers with 80-120 nm widths and clear edges, as examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The fiber formation is performed in a hierarchical manner: beta-sheet peptides form a protofibril, the protofibrils assemble side-by-side to form a ribbon, and the ribbons then coil in a left-handed fashion to make up a straight fiber. These type of fibers are formed from peptides possessing hydrophobic aromatic Phe residue(s). Furthermore, a peptide with Ala residues at both N and C termini does not form fibers (100 nm scale) with clear edges; this causes random aggregation of small pieces of fibers instead. Thus, the combination of unique amphiphilic sequences and terminal Pro residues determine the fiber morphology.     

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.     

Suppression of Oligomer Formation and Formation of Non‐Toxic Fibrils upon Addition of Mirror‐Image Aβ42 to the Natural l‐Enantiomer

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aβ) 42 is an aggregation‐prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror‐image d ‐Aβ42 should reduce the concentration of toxic oligomers formed from natural l ‐Aβ42. We synthesized l ‐ and D ‐Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l ‐Aβ42 at concentrations up to 50 μm . The mixing of Aβ42 enantiomers thus accelerates the formation of non‐toxic fibrils.     

Modulating artificial membrane morphology: pH-induced chromatic transition and nanostructural transformation of a bolaamphiphilic conjugated polymer from blue helical ribbons to red nanofibers.

Design and characterization of helical ribbon assemblies of a bolaamphiphilic conjugated polymer and their color-coded transformation into nanofibers are described. An L-glutamic acid modified bolaamphiphilic diacetylene lipid was synthesized and self-assembled into right-handed helical ribbons with micron scale length and nano scale thickness under mild conditions. The ribbon structures were further stabilized by polymerizing well-aligned diacetylene units to form bisfunctional polydiacetylenes (PDAs). Transitions from flat sheets to helical ribbons and tubes were observed by transmission electron microscopy. The helical ribbons appear to originate from the rupture of flat sheets along domain edges and the peeling off between stacked lipid layers. These results point to the applicability of chiral packing theory in bolaamphiphilic supramolecular assemblies. Contact mode atomic force microscopy observations revealed that high order existed in the surface packing arrangement. Hexagonal and pseudorectangular packings were observed in flat and twisted regions of the ribbons, respectively, suggesting a correlation between microscopic morphologies and nanoscopic packing arrangements. The tricarboxylate functionalities of the bolaamphiphilic lipid provide a handle for the manipulation of the bisfunctional PDAs' morphology. Increasing solution pH caused the fraying of helical ribbons into nanofibers accompanied by a sharp blue-to-red chromatic transition. A dramatic change in circular dichroism spectra was observed during this process, suggesting the loss of chirality in packing. A model is proposed to account for the pH-induced morphological change and chromatic transition. The color-coded transition between two distinct microstructures would be useful in the design of sensors and other "smart" nanomaterials requiring defined molecular templates.