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.     

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein–protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of “breathing motions” over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

Self-assembly of amylin(20-29) amide-bond derivatives into helical ribbons and peptide nanotubes rather than fibrils

Uncontrolled aggregation of proteins or polypeptides can be detrimental for normal cellular processes in healthy organisms. Proteins or polypeptides that form these amyloid deposits differ in their primary sequence but share a common structural motif: the (anti)parallel beta sheet. A well-accepted approach for interfering with beta-sheet formation is the design of soluble beta-sheet peptides to disrupt the hydrogen-bonding network; this ultimately leads to the disassembly of the aggregates or fibrils. Here, we describe the synthesis, spectroscopic analysis, and aggregation behavior, imaged by electron microscopy, of several backbone-modified amylin(20-29) derivatives. It was found that these amylin derivatives were not able to form fibrils and to some extent were able to inhibit fibril growth of native amylin(20-29). However, two of the amylin peptides were able to form large supramolecular assemblies, like helical ribbons and peptide nanotubes, in which beta-sheet formation was clearly absent. This was quite unexpected since these peptides have been designed as soluble beta-sheet breakers for disrupting the characteristic hydrogen-bonding network of (anti)parallel beta sheets. The increased hydrophobicity and the presence of essential amino acid side chains in the newly designed amylin(20-29) derivatives were found to be the driving force for self-assembly into helical ribbons and peptide nanotubes. This example of controlled and desired peptide aggregation may be a strong impetus for research on bionanomaterials in which special shapes and assemblies are the focus of interest.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Self-assembly and morphological characterization of two-component functional amyloid proteins

Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials. However, little has been known how different side domains, varied fusion positions and subunits affect self-assembly and morphologies of amyloid fibrils. Here, we constructed three groups of two-component amyloid proteins based on CsgA, the major protein components of Escherichia coli biofilms, to bridge these gaps. We showed that all fusion proteins have amyloid features, as indicated by Congo red assay. Atomic force microscopy (AFM) indeed reveals that these fusion proteins are able to self-assemble into fibrils, with an average diameter of 0.5-2 nm and length of hundreds of nanometers to several micrometers. The diameter of fibrils increases with the increase of the molecular weight of fusion domains, while the dynamic assembly of recombinant proteins was delayed as a result of the introduction of fusion domains. Moreover, fusion of the same functional domains but at intermediate position seems to cause the most interference on fibril assembly compared with those fused at C or Nterminus, as mainly short and irregular fibrils were detected. This phenomenon appears more pronounced for randomly coiled mussel foot proteins (Mfps) than for rigid chitin-binding domain (CBD). Finally, increase of the molecular weight of tandem repeats in protein monomer seemed to increase the fibril diameter of the resultant fibrils, but either reduction of the tandem repeats of CsgA to one single belta-sheet loop or increase in the number of tandem repeats of CsgAs from one to four produced shorter and intermittent fibrils compared with CsgA control protein. These studies therefore provide insights into self-assembly of two-component amyloid proteins and lay the foundation for rational design of multifunctional molecular biomaterials.     

Polymorphism of Amyloid Fibrils In Vivo

Polymorphism is a wide‐spread feature of amyloid‐like fibrils formed in vitro, but it has so far remained unclear whether the fibrils formed within a patient are also affected by this phenomenon. In this study we show that the amyloid fibrils within a diseased individual can vary considerably in their three‐dimensional architecture. We demonstrate this heterogeneity with amyloid fibrils deposited within different organs, formed from sequentially non‐homologous polypeptide chains and affecting human or animals. Irrespective of amyloid type or source, we found in vivo fibrils to be polymorphic. These data imply that the chemical principles of fibril assembly that lead to such polymorphism are fundamentally conserved in vivo and in vitro.     

Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation

Human calcitonin (hCT) is a 32-residue peptide hormone that can aggregate into amyloid fibrils and cause cellular toxicity. In this study, we investigated the inhibition effects of a group of polyphenolic molecules on hCT amyloid formation. Our results suggest that the gallate moiety in epigallocatechin-3-gallate (EGCG), a well-recognized amyloid inhibitor, is not critical for its inhibition function in the hCT amyloid formation. Our results demonstrate that flavonoid compounds, such as myricetin, quercetin, and baicalein, that contain vicinal hydroxyl groups on the phenyl ring effectively prevent hCT fibrillization. This structural feature may also be applied to non-flavonoid polyphenolic inhibitors. Moreover, our results indicate a plausible mechanistic role of these vicinal hydroxyl groups which might include the oxidation to form a quinone and the subsequent covalent linkage with amino acid residues such as lysine or histidine in hCT. This may further disrupt the crucial electrostatic and aromatic interactions involved in the process of hCT amyloid fibril formation. The inhibition activity of the polyphenolic compounds against hCT fibril formation may likely be attributed to a combination of factors such as covalent linkage formation, aromatic stacking, and hydrogen bonding interactions.     

Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.     

Quantitative prediction of amyloid fibril growth of short peptides from simulations: calculating association constants to dissect side chain importance

Quantitative prediction of the fibril growth properties of different peptides is conducted with a molecular dynamics approach. Association constants of small peptides used as a model for amyloid formation are calculated, and the results show very good agreement with experiments. Also the free-energy differences associated with two important interactions that characterize fibril growth, namely cross-beta-sheet and lateral interactions, are obtained. These two interactions show different dependencies on the physicochemical properties of the side chains, explaining the variation in fibril morphologies between different peptides.     

Formation of Mixed Ionic Complementary Peptide Fibrils

Summary: Two peptides (FEFEFKFK and HHHHHHFEFEFKFK) have been synthesised and their phase diagrams mapped out as a function of concentration and temperature. Both peptides formed self-supporting fibrillar hydrogels above a similar critical molar gelation concentration with a fibril diameter corresponding to the length of the fully stretched monomer. Mixing the peptides in a 9:1 ratio of FEFEFKFK to histadine functionalized FEFEFKFK in the presence of nanogold particles (binds to the histadine groups) resulted in thicker fibres suggesting that two fibrils associate together to form a fibre. TEM studies revealed that the gold particles were distributed throughout the hydrogel and adjacent to both sides of the fibrillar structures with an average distance between particles of 21 nm. It is postulated that the peptides form anti-parallel beta sheet fibrils that associate together via π-stacking interactions between the imidazole side chains of the histadine groups to form a fibre, where on average 1 in every 44 peptides is functionalized.     

Utilizing Lifetimes to Suppress Random Coil Features in 2D IR Spectra of Peptides

We report that the waiting time delay in 2D IR pulse sequences can be used to suppress signals from structurally disordered regions of amyloid fibrils. At a waiting time delay of 1.0 ps, the random coil vibrational modes of amylin fibrils are no longer detectable, leaving only the sharp excitonic vibrational features of the fibril β-sheets. Isotope labeling with (13)C(18)O reveals that structurally disordered residues decay faster than residues protected from solvent. Since structural disorder is usually accompanied by hydration, we conclude that the shorter lifetimes of random-coil residues is due to solvent exposure. These results indicate that 2D IR pulse sequences can utilize the waiting time to better resolve solvent-protected regions of peptides and that local mode lifetimes should be included in simulations of 2D IR spectra.     

Capturing intermediate structures of Alzheimer's beta-amyloid, Abeta(1-40), by solid-state NMR spectroscopy

Molecular structures of diffusible amyloid intermediates, commonly observed in misfolding of amyloid proteins into fibrils, have attracted broad interest because the intermediates may be potent neurotoxins responsible for amyloid diseases such as Alzheimer's disease (AD) and because the intermediate structures provide an experimental basis for defining the misfolding pathway. However, owing to the intrinsically unstable and noncrystalline nature of the systems, traditional approaches such as X-ray crystallography and solution NMR have been ineffective for elucidating molecular-level structures of the amyloid intermediates. We present a novel approach using solid-state NMR (SSNMR) that permitted the first site-resolved structural measurement of an intermediate species in fibril formation for a 40-residue Alzheimer's beta-amyloid peptide, Abeta(1-40). In this approach, we combined detection of conformation and morphology changes by fluorescence spectroscopy and electron microscopy and quantitative structural examination for freeze-trapped intermediates by SSNMR. The results provide the initial evidence that a spherical amyloid intermediate of 15-30 nm in diameter exists prior to fibril formation of Abeta(1-40) and that the intermediate involves well-ordered beta-sheets in the C-terminal and hydrophobic core regions. The SSNMR-based approach presented here could be applied to intermediate species of diverse amyloid proteins.     

Peptide‐binding specificity of the prosurfactant protein C Brichos domain analyzed by electrospray ionization mass spectrometry

The C‐terminal domain of lung surfactant protein C (CTC) precursor (proSP‐C) is involved in folding of the transmembrane segment of proSP‐C. CTC includes a Brichos domain with homologs in cancer‐ and dementia‐associated proteins. Mutations in the Brichos domain cause misfolding of proSP‐C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI‐MS) with collision‐induced dissociation (CID) experiments was applied to study non‐covalent interactions between human recombinant CTC or its Brichos domain, and SP‐C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide‐binding function of CTC. In titration experiments, apparent dissociation constants (K_D) were in the micromolar range where triple‐valine showed the lowest K_D and triple‐tyrosine the highest. Non‐hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non‐aromatic ligands. The Brichos domain was also shown to bind fibril‐forming peptides containing aromatic/hydrophobic residues. Copyright © 2009 John Wiley & Sons, Ltd.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

Solid-state NMR detection of 14N-13C dipolar couplings between amino acid side groups provides constraints on amyloid fibril architecture

Solid‐state nuclear magnetic resonance (SSNMR) is a powerful technique for the structural analysis of amyloid fibrils. With suitable isotope labelling patterns, SSNMR can provide constraints on the secondary structure, alignment and registration of β‐strands within amyloid fibrils and identify the tertiary and quaternary contacts defining the packing of the β‐sheet layers. Detection of ¹⁴N? ¹³C dipolar couplings may provide potentially useful additional structural constraints on β‐sheet packing within amyloid fibrils but has not until now been exploited for this purpose. Here a frequency‐selective, transfer of population in double resonance SSNMR experiment is used to detect a weak ¹⁴N? ¹³C dipolar coupling in amyloid‐like fibrils of the peptide H₂N‐SNNFGAILSS‐COOH, which was uniformly ¹³C and ¹⁵N labelled across the four C‐terminal amino acids. The ¹⁴N? ¹³C interatomic distance between leucine and asparagine side groups is constrained between 2.4 and 3.8 Å, which allows current structural models of the β‐spine arrangement within the fibrils to be refined. This procedure could be useful for the general structural analysis of other proteins in condensed phases and environments, such as biological membranes. Copyright © 2011 John Wiley & Sons, Ltd.     

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

A number of proteins are capable of converting from their soluble, monomeric form into highly-ordered, insoluble aggregates known as amyloid fibrils. In vivo, these fibrils, which accumulate in organs and tissues, are associated with a wide range of amyloid diseases for which there are currently no therapeutic solutions. The molecular details of the pathway from native monomer through oligomeric intermediates to the final amyloid fibril remain a challenging enigma. Over the past few years, mass spectrometry has been applied to investigate the various stages of amyloid fibril formation, and this report summarizes the key steps achieved to date.     

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-β Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation

Alzheimer’s disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-β (Aβ) peptides. This review article presents molecular dynamics (MD) simulation studies of Aβ peptides and Aβ fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aβ, a β-hairpin structure promotes the formation of intermolecular β-sheet structures. Aβ peptides tend to exist at hydrophilic/hydrophobic interfaces and form more β-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aβ peptides, interact with Aβ. An MD simulation study of the Aβ amyloid fibrils in equilibrium is also presented: the Aβ amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer’s disease using MD simulations.     

Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly: implications for amyloid fibril formation and materials science

Deciphering the mechanism(s) of β-sheet mediated self-assembly is essential for understanding amyloid fibril formation and for the fabrication of polypeptide materials. Herein, we report a simple peptidomimetic that self-assembles into polymorphic β-sheet quaternary structures including protofilaments, filaments, fibrils, and ribbons that are reminiscent of the highly ordered structures displayed by the amyloidogenic peptides Aβ, calcitonin, and amylin. The distribution of quaternary structures can be controlled by and in some cases specified by manipulating the pH, buffer composition, and the ionic strength. The ability to control β-sheet-mediated assembly takes advantage of quaternary structure dependent pK(a) perturbations. Biophysical methods including analytical ultracentrifugation studies as well as far-UV circular dichroism and FT-IR spectroscopy demonstrate that linked secondary and quaternary structural changes mediate peptidomimetic self-assembly. Electron and atomic force microscopy reveal that peptidomimetic assembly involves numerous quaternary structural intermediates that appear to self-assemble in a convergent fashion affording quaternary structures of increasing complexity. The ability to control the assembly pathway(s) and the final quaternary structure(s) afforded should prove to be particularly useful in deciphering the quaternary structural requirements for amyloid fibril formation and for the construction of noncovalent macromolecular structures.     

Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.