We present a structural model for amyloid fibrils formed by the 40-residue ␤-amyloid peptide associated with Alzheimer's disease (A␤ 1-40), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-␤ structural motif established by x-ray fiber diffraction and satisfies constraints on A␤ 1-40 fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of A␤ 1-40 are structurally disordered in the fibrils. Residues 12-24 and 30 -40 adopt ␤-strand conformations and form parallel ␤-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two ␤-sheets in contact through sidechain-sidechain interactions. A single cross-␤ unit is then a double-layered ␤-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-␤ units with their hydrophobic faces juxtaposed.A myloid fibrils are filamentous structures, with typical diameters of Ϸ10 nm and lengths up to several micrometers, formed by numerous peptides and proteins with disparate sequences and molecular weights. Biomedical interest in amyloid fibrils arises from their occurrence in amyloid diseases (1), including Alzheimer's disease, type 2 diabetes, Huntington's disease, and prion diseases. Current interest in the molecular structures of amyloid fibrils additionally arises from fundamental questions regarding the molecular mechanism of amyloid formation and the nature of the intermolecular interactions that stabilize these structures for an extremely diverse class of polypeptides.No high-resolution molecular structure of an amyloid fibril has yet been determined experimentally because amyloid fibrils are noncrystalline solid materials and are therefore incompatible with x-ray crystallography and liquid state NMR. X-ray fiber diffraction shows that amyloid fibrils contain cross-␤ structural motifs, i.e., extended ␤-sheets in which the ␤-strand segments run approximately perpendicular to, and the intermolecular hydrogen bonds run approximately parallel to, the long axis of the fibril (2, 3). Other molecular-level structural features of amyloid fibrils are not well established.In the case of fibrils formed by the full-length ␤-amyloid peptide associated with Alzheimer's disease (A␤), which ranges from 39 to 43 residues in length in vivo (4, 5), several molecular models have been proposed (6-10). These models exhibit many qualitative and quantitative differences, reflecting the paucity of experimental constraints. All of these models are inconsistent with recent measurements of 13 C-13 C nuclear magnetic dipole-dipole couplings (i.e., intermolecular distances) by solid state NMR (11-13), which imply an in-register parallel alignment of peptide chains within the cross-␤ motif in A␤ 1-40 and A␤ 1-42 fibrils (A␤ mϪn denotes residues m t...

The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH(2), called A beta(16-22) and representing residues 16-22 of the full-length beta-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in A beta(16-22) fibrils are investigated by solid state (13)C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a beta-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit (13)C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional (13)C-(13)C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain (13)C NMR signals and indicates that the beta-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. (13)C multiple-quantum (MQ) NMR and (13)C/(15)N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of beta-sheets in the A beta(16-22) fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of beta-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. A beta(16-22) is among the shortest fibril-forming fragments of full-length beta-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.

Senile plaques associated with Alzheimer's disease contain deposits of fibrils formed by 39-to 43-residue ␤-amyloid peptides with possible neurotoxic effects. X-ray diffraction measurements on oriented fibril bundles have indicated an extended ␤-sheet structure for Alzheimer's ␤-amyloid fibrils and other amyloid fibrils, but the supramolecular organization of the ␤-sheets and other structural details are not well established because of the intrinsically noncrystalline, insoluble nature of amyloid fibrils. Here we report solid-state NMR measurements, using a multiple quantum (MQ) 13 C NMR technique, that probe the ␤-sheet organization in fibrils formed by the full-length, 40-residue ␤-amyloid peptide (A␤1-40). Although an antiparallel ␤-sheet organization often is assumed and is invoked in recent structural models for full-length ␤-amyloid fibrils, the MQNMR data indicate an in-register, parallel organization. This work provides site-specific, atomic-level structural constraints on full-length ␤-amyloid fibrils and applies MQNMR to a significant problem in structural biology.A variety of peptides and proteins form amyloid fibrils, including those involved in amyloid diseases such as Alzheimer's disease, type II diabetes, and spongiform encephalopathies (1, 2), as well as proteins that are studied primarily as models for the elucidation of fundamental aspects of protein biophysics (3, 4). Compared with monomeric or oligomeric forms of peptides and proteins, relatively little is known definitively about the molecular structure and supramolecular organization of amyloid fibrils. All amyloid fibrils exhibit similar morphologies in electron micrographs (Fig. 1A), despite their diversity of amino acid sequences and sequence lengths. X-ray diffraction measurements on oriented amyloid fibril bundles show a characteristic ''cross-␤'' pattern (2, 5-8), implying an extended ␤-sheet structure with polypeptide chains running roughly perpendicular to the long axis of the fibril and interchain hydrogen bonds roughly parallel to this axis. This ␤-sheet structure is supported by recent cryo-electron microscopy measurements (9). Finer structural details are generally not well established because amyloid fibrils are not amenable to structural characterization by single-crystal diffraction and highresolution, liquid-state NMR techniques.Amyloid fibrils that form senile plaques in Alzheimer's disease are comprised of ␤-amyloid (A␤) peptides that range in length from 39 to 43 residues (10-12). Various shorter fragments of the full-length A␤ peptides are also known to fibrillize in vitro (13)(14)(15)(16)(17)(18)(19)(20)(21). A number of structural models for full-length A␤ fibrils (8,(22)(23)(24)(25) and fibrils formed by A␤ fragments (13-16, 21) have been proposed, most (16,(21)(22)(23)(24)(25)(26), but not all (13-15), of which are based on an antiparallel ␤-sheet organization. Here we present solid-state 13 C NMR data, obtained with multiple quantum (MQ) NMR techniques based on time-reversal principles (27-32), which support an exte...

We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (A beta(1-40)) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between (13)C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional (13)C magic-angle spinning NMR spectra of the labeled A beta(1-40) samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 +/- 0.5 A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly beta-sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A beta(1-40), including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length beta-amyloid fibrils.

No abstract

We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1)(-)(42)) and by residues 10-35 of the full-length peptide (Abeta(10)(-)(35)). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of beta-sheets present in the characteristic amyloid cross-beta structural motif and the assembly of these beta-sheets into a fibril. EM images of negatively stained Abeta(10)(-)(35) fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Abeta(10)(-)(35) fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-beta layer. Abeta(10)(-)(35) fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Abeta(1)(-)(42) fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-beta layer are also formed by Abeta(1)(-)(42) and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Abeta(10)(-)(35) fibrils, using multiple-quantum (13)C NMR, (13)C-(13)C dipolar recoupling, and (15)N-(13)C dipolar recoupling techniques, support the in-register parallel beta-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this beta-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Abeta(1)(-)(42) fibrils, which represent the first NMR data on Abeta(1)(-)(42) fibrils, also indicate an in-register parallel beta-sheet organization. These results, along with previously reported data on Abeta(1)(-)(40) fibrils, suggest that the supramolecular structures of Abeta(10)(-)(35), Abeta(1)(-)(40), and Abeta(1)(-)(42) fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.

Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40 . Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40 .

We report on the synthesis, characterisation, and physical and tribological properties of halogen-free ionic liquids based on various chelated orthoborate anions with different phosphonium cations, both without halogen atoms in their structure. Important physical properties of the ILs including glass transition temperatures, density, viscosity and ionic conductivity were measured and are reported here. All of these new halogen-free orthoborate ionic liquids (hf-BILs) are hydrophobic and hydrolytically stable liquids at room temperature. As lubricants, these hf-BILs exhibit considerably better antiwear and friction reducing properties under boundary lubrication conditions for steel-aluminium contacts as compared with fully formulated (15W-50 grade) engine oil. Being halogen free these hf-BILs offer a more environmentally benign alternative to ILs being currently developed for lubricant applications.