Structural and Dynamic Features of Alzheimer's Aβ Peptide in Amyloid Fibrils Studied by Site-directed Spin Labeling

Abstract

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid ␤ (A␤) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, A␤40 and A␤42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.Protein deposits are commonly associated with a wide range of degenerative diseases, including Alzheimer's disease, Parkinson's disease, type-2 diabetes, macular degeneration, and several others. These deposits contain a number of proteins that often have fibrillar morphology. In Alzheimer's disease, the extracellular deposits are largely made up of a short 39 -42-amino-acid-long peptide referred to as A␤.Like all other amyloid fibrils, A␤ fibrils are thought to have a cross ␤-structure, in which individual ␤-strands are organized roughly perpendicular to the fiber axis. According to fiber x-ray diffraction studies of A␤ fibrils (1, 2) the unit spacing between these individual ␤-strands along the fibril axis is 4.7 Å, while the sheets are spaced at about 10 Å. However, our understanding of the exact structure of A␤ or that of other amyloid fibrils is still very incomplete. For example, it is not known which regions of the peptide are directly involved in the formation of the cross ␤-structure. Simple geometric considerations suggest that not all amino acids in A␤40 or A␤42 can be part of the cross ␤-structure in a linear, fully extended ␤-strand. Assuming an average distance between residues in an extended ␤-strand of 3.5 Å per residue, a completely extended structure model predicts a minimum extended length of 140 Å for A␤40. This length is considerably larger than the diameter of the putative building blocks of the fibrils, protofilaments with an individual diameter of 40 -55 Å (3-6). In fact, it even exceeds the 60 -100-Å diameter of the entire fibril. Thus, turn or bend regions are needed to account for the fiber dimensions. Indeed, hydrogen-deuterium exchange measurements of fibrillar A␤ (7) suggest that only about half of the peptide is involved in a protective hydrogen-bonded structure. In addition, Fourier transform infrared spectroscopic studies on fibrils indicate the presence of at least one turn (8, 9). However, the locations of these turns or the location of the ␤-strands are still unclear.Furthermore, there has also been some controversy regarding the arrangement of individual peptides with respect to each other. Fourier transform infrared spectroscopic data indicate an antiparallel organization of the ␤-sheets within the A␤ fibril (8,10). An antiparallel organization is al...

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

“…Although these spectra are somewhat less unambiguous in their interpretation, they are certainly not inconsistent with our proposed parallel, in-register packing motif of the PrP amyloid. Similar EPR signals have been observed previously for spinlabeled A␤ fibrils (30), and these amyloid structures were shown by NMR to adopt parallel and in-register alignment of ␤-strands (31,32). An intrinsic caveat of all spin-labeling studies of proteins relates to the presence of a relatively bulky nitroxide label that can (and often does) introduce local structural perturbation, with a degree of this perturbation depending largely on steric interactions with neighboring side chains.…”

“…Previous attempts in this regard were limited to global topological studies by using digital reconstitution of electron micrographs of fibrils formed from PrP monomers lacking the native disulfide bond (33). One of few techniques suitable for a higher resolution analysis is site-directed spin labeling (26,27,30). Using this approach, here we have determined that the core of the PrP amyloid is located within the C-terminal region encompassing residues Ϸ160-220.…”

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)(35)(36), electron microscopy (37)(38)(39)(40)(41)(42)(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 …”

Amyloid and Alzheimer’s disease

“…From structural studies by solid-state NMR (nuclear magnetic resonance) (Petkova et al, 2002), site-directed spin-labeling EPR (electron paramagnetic resonance) ( Török et al, 2002), and hydrogen/deuterium-exchange (HX) ( Lührs et al, 2005), it was revealed that Aβ can form a protofibril structure by stacking hairpin-like building blocks and forming a two-layered β-sheet (Fig. 1).…”