The ultimate goal of structural studies of proteins is to gain insight into protein three-dimensional structure at highresolution level. This can often be accomplished by the application of techniques such as X-ray crystallography or multidimensional nuclear magnetic resonance (NMR) spectroscopy. However, high-resolution studies of proteins are not always feasible. For example, crystallographic studies require high-quality single crystals which for many proteins (e.g., the vast majority of membrane proteins) are not available. Furthermore, the question arises as to whether the relatively "static" structure in single crystals adequately represents the protein conformation in a complex and dynamic environment of living cells. There is a growing realization [e.g., Martinek et al. (1989)] that in vivo most proteins act in an interfacial environment where they form dynamic complexes with biological membranes, nucleic acids, polysaccharides, or other proteins. Aqueous buffers, from which protein crystals are usually grown, do not necessarily mimic well the conditions of protein functioning in vivo. NMR offers a somewhat better flexibility in studying protein structure in "biologically relevant" environments. However, the interpretation of NMR spectra of larger proteins is very complex, and the assignment of interproton distances generated by the NMR experiment is not always feasible; at present the technique is restricted to small proteins of less than approximately 15-20 kDa.The practical limitations encountered in high-resolution structural studies of proteins stimulate continual progress in f This is National Research Council of Canada Publication No. NRCC 34263.

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To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimer's disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and ␣-helical prion protein, PrP C , to the ␤-sheet-rich PrP Sc . This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP C monomer, structures of the pathogenic PrP Sc or synthetic PrP Sc -like aggregates remain elusive. Here we have used sitedirected spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP C . Our data show that, in contrast to earlier, largely theoretical models, the conformational conversion of PrP C involves major refolding of the C-terminal ␣-helical region. The core of the amyloid maps to C-terminal residues from Ϸ160 -220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of ␤-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to ␤-structure.EPR ͉ spin labeling ͉ transmissible spongiform encephalopathy

A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by 13 C, 15 N-labeled huPrP23-144. Surprisingly, we find that signals from Ϸ100 residues (i.e., Ϸ80% of the sequence) are not detected above approximately ؊20°C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112-141. These resonances are remarkably narrow, exhibiting average 13 C and 15 N linewidths of Ϸ0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23-144 amyloid encompassing residues 112-141. Analysis of 13 C secondary chemical shifts identified likely ␤-strand segments within this core region, including ␤-strand 130 -139 containing critical residues 138 and 139. In contrast to this relatively rigid, ␤-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.protein structure ͉ solid-state NMR ͉ protein misfolding ͉ transmissible spongiform encephalopathies

Soluble oligomers of A␤42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP C , on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128 -1132). Here we characterized the interaction between synthetic A␤42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met 129 and Val 129 polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with A␤ oligomers. It was also found that in addition to the previously identified ϳ95-110 segment, the second region of critical importance for the interaction with A␤42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for A␤42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well. Alzheimer disease (AD)2 is a devastating age-related neurodegenerative disorder leading to memory loss and progressive decline in cognitive ability (1-3). Pathologically, the disease is characterized by the formation of neurofibrillary tangles composed of hyperphosphorylated Tau and accumulation of extracellular amyloid plaques (1-6). The main component of these plagues is the 40 -42-amino acid residue amyloid-␤ (A␤) peptide, a product of proteolytic processing of a large membrane glycoprotein, amyloid precursor protein (APP).Although the etiology of AD remains poorly understood, the leading hypothesis is that the major causative agent is the aggregated form of A␤ (4, 7). Although in the past much attention has focused on mature ␤-sheet-rich amyloid fibrils, more recent evidence points to a critical role of smaller, soluble A␤ oligomers (8 -13). In contrast to poor quantitative correlation between the burden of insoluble fibrillar amyloid plagues and the degree of dementia, the severity of AD appears to correlate well with the concentration of soluble A␤ oligomers (9 -11). Furthermore, these soluble oligomers have been shown to be potent neurotoxins in vitro and in vivo; among other effects, they were reported to inhibit hippocampal long term potentiation, a widely used electrophysiological measure of synaptic plasticity related to learning and memory, and cause impairment of long term memory in rats (...

Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrP C , into a misfolded, oligomeric form, PrP Sc . Although the high-resolution structure of the PrP C is well characterized, the structural properties of PrP Sc remain elusive. Here we used MS analysis of H/D backbone amide exchange to examine the structure of amyloid fibrils formed by the recombinant human PrP corresponding to residues 90 -231 (PrP90 -231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrP C . Analysis of H/D exchange data allowed us to map the systematically H-bonded ␤-sheet core of PrP amyloid to the C-terminal region (staring at residue Ϸ169) that in the native structure of PrP monomer corresponds to ␣-helix 2, a major part of ␣-helix 3, and the loop between these two helices. No extensive hydrogen bonding (as indicated by the lack of significant protection of amide hydrogens) was detected in the N-terminal part of PrP90 -231 fibrils, arguing against the involvement of residues within this region in stable ␤-structure. These data provide long-sought experimentally derived constraints for high-resolution structural models of PrP amyloid fibrils.prion diseases ͉ transmissible spongiform encephalopathy ͉ amyloid structure ͉ mass spectrometry

Spongiform encephalopathies are believed to be transmitted by self-perpetuating conformational conversion of the prion protein. It was shown recently that fundamental aspects of mammalian prion propagation can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop (PrP23-144). Here we demonstrate that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that these structural differences are controlled by one or two residues in a critical region. These sequence-specific structural characteristics correlate strictly with the seeding specificity of amyloid fibrils. However, cross-seeding of PrP23-144 from one species with preformed fibrils from another species may overcome natural sequence-based structural preferences, resulting in a new amyloid strain that inherits the secondary structure and morphology of the template. These data provide direct biophysical evidence that protein conformations are transmitted in PrP amyloid strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.