Probing solvent accessibility of amyloid fibrils by solution NMR spectroscopy

Abstract

Amyloid is the result of an anomalous protein and peptide aggregation, leading to the formation of insoluble fibril deposits. At present, 18 human diseases have been associated with amyloid deposits-e.g., Alzheimer's disease and Prion-transmissible Spongiform Encephalopathies. The molecular structure of amyloid is to a large extent unknown, because of lack of high-resolution structural information within the amyloid state. However, from other experimental data it has been established that amyloid fibrils predominantly consist of ␤-strands arranged perpendicular to the fibril axis. Identification of residues involved in these secondary structural elements is therefore of vital importance to rationally designing appropriate inhibitors. We have designed a hydrogen͞ deuterium exchange NMR experiment that can be applied on mature amyloid to enable identification of the residues located inside the fibril core. Using a highly amyloidogenic peptide, corresponding to residues 25-35 within the Alzheimer A␤(1-43) peptide, we could establish that residues 28 -35 constitute the amyloid core, with residues 31 and 32 being the most protected. In addition, quantitative values for the solvent accessibility for each involved residue could be obtained. Based on our data, two models of peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors.A myloid diseases have in common an abnormal folding of normally soluble proteins resulting in the formation of extracellular amyloid deposits (1). Examples are Alzheimer's Disease, Prion-transmissible Spongiform Encephalopathies, and Familial Amyloidotic Polyneuropathy (2). Through selfassembly these proteins produce regular fibrillar structures possessing a predominant ␤-sheet conformation (3). Structural information on the core of fibrils has mainly been elucidated from fiber-diffraction studies (4), mass-spectrometry (5), and solid-state NMR spectroscopy (6), resulting in a general picture consistent with parallel or antiparallel ␤-strands, placed perpendicularly to the fibril axis. However, a detailed structure of a fibril at the atomic level is still lacking. In this respect, more structural information-e.g., which specific amino acids make up the amyloid-forming core-becomes crucially important.In this article, we describe a NMR approach to determining the sequence-specific structural elements in the fibril stage of amyloid forming proteins. The method relies on the partial solvent protection of hydrogen-bonded amide protons connecting the ␤-strands throughout the length of the fibril. In aqueous solutions it is expected that amide protons located on the exterior of the fibril are more accessible to solvent and therefore experience a higher hydrogen exchange rate than amide protons buried within the fibril interior. Studies using mass spectrometry, in combination with deuterium exchange, have already pointed to this possibility (7). However, mass spectrometry does not allow spe...

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

“…From the standpoint of molecular structure, the defining feature of an amyloid fibril is the presence of cross-β supramolecular structure, meaning that the β-sheets within the fibril are formed by β-strand segments that run approximately perpendicular to the long axis of the fibril and are linked by hydrogen bonds that run approximately parallel to this axis (11)(12)(13). Although determination of the molecular structures of amyloid fibrils is made difficult by their inherent noncrystallinity and insolubility, techniques such as solid state nuclear magnetic resonance (NMR) (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), electron paramagnetic resonance (EPR) (34-36), electron microscopy (37-43), hydrogen/deuterium exchange (29,(44)(45)(46)(47), scanning mutagenesis (48), chemical crosslinking (27,49,50), and x-ray diffraction of amyloid-like crystals (51,52) have recently shed substantial light on these structures.In vitro, amylin readily forms amyloid fibrils with a variety of morphologies as seen in transmission electron microscope (TEM) and atomic force microscope (AFM) images (40,41), and with typical cross-β diffraction patterns (53). Early solid state NMR studies by Griffiths et al focused on fibrils formed by a peptide representing residues 20-29 of amylin.…”

Probing Solvent Accessibility of Transthyretin Amyloid by Solution NMR Spectroscopy

“…We have recently developed a general method using NMR spectroscopy in combination with hydrogen/deuterium (H/D) exchange to facilitate the identification of individual core residues within a fibril (10). The technique relies on the partial solvent protection of hydrogenbonded amide protons throughout the length of the fibril.…”

Modular peptides from the thermoplastic squid sucker ring teeth form amyloid-like cross-β supramolecular networks

“…The exchange rates and protection factors of the fibers are comparable to those of robust Aβ (1-42) and Aβ (25-35) amyloid fibrils. [53,57] While the H/D exchange for all residues generally increased with temperature, we note an abnormal behaviour at 45ºC, where all residues not only exhibited a higher exchange than at 65ºC but also a large fluctuation in peak intensity, which is evidenced by the large error bars. It is important to note that such high standard deviations appear to be especially prominent at 45ºC and not at other temperatures, indicating that it represents an intrinsic feature of the peptide but is not due to experimental uncertainties.…”

“…H/D exchange NMR studies denote strong protection factors for residues in A2 microfibers ( Figure 6B) comparable to amyloid peptides. [53,57] However, residues in A1 microfibers ( Figure 6D) exhibit even higher protection factors. These observations suggest that the presence of Pro allows the cross β-structure packing to relax by extending the hydrogen bond lengths, [84] possibly preventing premature aggregation and in turn ensuring further assembly of the supramolecular network, which could be consistent with the higher amount of β-sheet content predicted by MD simulations for the Pro-containing A2 peptide at equilibrium.…”

“…[52] In order to gain a deeper understanding of the stability of residues involved in stabilizing these β-sheet rich fibrils, we subjected the microfibers to hydrogendeuterium (H/D) exchange and NMR spectroscopy, according to a method previously developed to identify residues constituting the amyloid core. [53] In brief, the method identifies the core residues involved in hydrogen bonding by monitoring which backbone amides of the β-sheet stabilized fibrils are the most susceptible to H-bonding disruption. Accordingly, when the fibrils are exposed to deuterium, the labile backbone amide protons exchange with deuterium at a rate controlled by the extent of solvent exposure.…”