Solid-state NMR characterization of Alzheimer-like tau amyloid fibrils.

Eines der bedeutendsten Kennzeichen des Morbus Alzheimer ist die Zusammenlagerung des Mikrotubuli-assoziierten Proteins Tau in Fibrillen, die als „gepaarte helikale Filamente“ (PHF) bezeichnet werden. Allerdings sind die strukturellen Grundlagen der PHF Aggregation auf atomarer Ebene weitestgehend unbekannt. In dieser Studie wurden mittels Festkörper-Kernspinresonanz-Spektroskopie (FK-NMR) in vitro hergestellte PHF einer Tau Isoform untersucht, die aus drei Wiederholungseinheiten besteht und den Kern der PHF repräsentiert (K19). Wir haben herausgefunden, dass der rigide Kern der Fibrillen von den Aminosäuren V306 bis S324 – lediglich 18 von 99 Residuen – gebildet wird und aus 3 β-Faltblatt-Strängen besteht, die durch zwei kurze Knickstellen miteinander verbunden sind. Der erste β-Strang wird von dem gut untersuchten Hexapeptid 306VQIVYK311 gebildet. Von diesem ist bekannt, dass es sich ebenfalls zusammenlagern kann und dabei so genannte hydrophobe „steric zipper“ Kontakte ausbildet. Ergebnisse an einer gemischt [15N:13C]-markierten K19 PHF Probe zeigen, dass sich die β-Stränge parallel und nicht zu einander verschoben übereinander lagern. Zwischen C322-Resten verschiedener Moleküle bilden sich Disulfid-Brücken (DSB) aus, die zu einer lokalen Beeinträchtigung der β-Faltblatt-Struktur führen, wodurch in den FK-NMR Spektren Polymorphismus beobachtbar ist. Insbesondere die Aminosäurereste K321-S324 weisen zwei Resonanz-Sätze auf. Des Weiteren bestätigen Experimente, die an K19 C322A PHF durchgeführt wurden, den Einfluss der DSB auf die Struktur des Fibrillenkerns. Die Strukturdaten werden durch H/D-Austausch NMR Messungen an K19 sowie K18, einer Isoform bestehend aus vier Wiederholungseinheiten, gestützt. Zielgerichtete Mutagenese-Studien an K19 zeigen, dass Mutationen innerhalb der drei verschiedenen β-Stränge zu einem signifikanten Verlust der PHF Aggregationseffizienz führen, was die Bedeutung der β-Strang-reichen Region für die Zusammenlagerung von Tau Proteinen unterstreicht

Spontaneous aggregation of the insulin-derived steric zipper peptide VEALYL results in different aggregation forms with common features.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-beta spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite,for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [C-13,N-15]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the beta-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as empty set and Psi backbone torsion angles calculated with TALOS, are indicative of beta-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved

Transthyretin Aggregation Pathway toward the Formation of Distinct Cytotoxic Oligomers

Characterization of small oligomers formed at an early stage of amyloid formation is critical to understanding molecular mechanism of pathogenic aggregation process. Here we identifed and characterized cytotoxic oligomeric intermediates populated during transthyretin (TTR) aggregation process. Under the amyloid-forming conditions, TTR initially forms a dimer through interactions between outer strands. The dimers are then associated to form a hexamer with a spherical shape, which serves as a building block to self-assemble into cytotoxic oligomers. Notably, wild-type (WT) TTR tends to form linear oligomers, while aTTR variant(G53A) prefers forming annular oligomers with pore-like structures. Structural analyses of the amyloidogenic intermediates using circular dichroism (CD) and solid-state NMR revealthatthe dimer and oligomers have a signifcant degree of native-like β-sheet structures (35–38%), but with more disordered regions (~60%)than those of nativeTTR.TheTTR variant oligomers are also less structured than WT oligomers. The partially folded nature of the oligomeric intermediates might be a common structural property of cytotoxic oligomers.The higher fexibility of the dimer and oligomers may also compensate for the entropic loss due to the oligomerization of the monomers

Factors That Drive Peptide Assembly and Fibril Formation: Experimental and Theoretical Analysis of Sup35 NNQQNY Mutants

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single β-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early β-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms

Spontaneous aggregation of the insulin-derived steric zipper peptide VEALYL results in different aggregation forms with common features.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-beta spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite,for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [C-13,N-15]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the beta-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as empty set and Psi backbone torsion angles calculated with TALOS, are indicative of beta-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved