One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three β-strands connected by two short kinks. The first β-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that β-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the β-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different β-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the β-structure-rich regions for tau aggregation.

Roll out the barrel: The conformation of the N‐terminal domain of a functional human voltage‐dependent anion channel (hVDAC1) in lipid bilayers has been determined (see picture; overlay of NMR model (blue) and X‐ray structure (red/gray)). Solid‐state NMR spectroscopy reveals that the N terminus assumes a well‐defined, rigid structure and that its removal induces a conformational change in the hVDAC1 β‐barrel.

The process of protein misfolding and self-assembly into various, polymorphic aggregates is associated with a number of important neurodegenerative diseases. Only recently, crystal structures of several short peptides have provided detailed structural insights into -sheet rich aggregates, known as amyloid fibrils. Knowledge about early events of the formation and interconversion of small oligomeric states, an inevitable step in the cascade of peptide self-assembly, however, remains still limited. We employ molecular dynamics simulations in explicit solvent to study the spontaneous aggregation process of steric zipper peptide segments from the tau protein and insulin in atomistic detail. Starting from separated chains with random conformations, we find a rapid formation of structurally heterogeneous, -sheet rich oligomers, emerging from multiple bimolecular association steps and diverse assembly pathways. Furthermore, our study provides evidence that aggregate intermediates as small as dimers can be kinetically trapped and thus affect the structural evolution of larger oligomers. Alternative aggregate structures are found for both peptide sequences in the different independent simulations, some of which feature characteristics of the known steric zipper conformation (e.g., -sheet bilayers with a dry interface). The final aggregates interconvert with topologically distinct oligomeric states exclusively via internal rearrangements. The peptide oligomerization was analyzed through the perspective of a minimal oligomer, i.e., the dimer. Thereby all observed multimeric aggregates can be consistently mapped onto a space of reduced dimensionality. This novel method of conformational mapping reveals heterogeneous association and reorganization dynamics that are governed by the characteristics of peptide sequence and oligomer size.

Co-translational protein folding is not yet well understood despite the availability of high-resolution ribosome crystal structures. We present first solid-state NMR data on non-mobile regions of a prokaryotic ribosomal complex. Localized chemical shift perturbations and line broadening are observed for the backbone amide resonances corresponding to the regions in the trigger factor ribosome-binding domain that are involved in direct contact with the ribosome or undergo conformational changes upon ribosome binding. This large asymmetric protein complex (1.4 MDa) becomes accessible for NMR investigations by the combined use of proton detection and high MAS frequencies (60 kHz). The presented results open new perspectives for the understanding of the mechanism of large molecular machineries.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-β 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) [(13)C,(15)N]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 β-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 Ø and ψ backbone torsion angles calculated with TALOS, are indicative of β-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.

New derivatives of pyrazolate-based binucleating ligands HL with appended imine functions have been synthesized to provide a versatile set of ligand systems with different backbone substituents both at the pyrazole-C(4) and the imine-C (H, Me, Ph). These scaffolds have two adjacent coordination compartments akin to the alpha-diimine type. A series of binuclear palladium(II) complexes [LPd(2)Cl(3)] (1-4) and tetranuclear nickel(II) complexes [L(2)Ni(4)Br(6)(solvent)(4)] (5, 6) of the various ligands have been prepared and characterized, including X-ray structural analyses for two representative Pd and the two Ni complexes. Complexes 5 and 6 were found to contain an unusual central mu(4)-bromide. Mononuclear nickel(II) complexes [L(2)Ni] were detected as intermediates in the formation of the tetranuclear complexes and have been characterized by X-ray analyses in two cases (7, 8). The interconversion between 5' and 7 has been investigated by UV/Vis spectroscopy and ESI mass spectrometry, and magnetic coupling in the [L(2)Ni(4)Br(6)(solvent)(4)] complexes has been studied (SQUID). Trans-coupling via the central mu(4)-bromide is suggested to mediate significant antiferromagnetic interaction. The reactivity of such types of Pd and Ni complexes has been tested for the vinyl/addition polymerization of norbornene. In the presence of an excess of cocatalyst methylaluminoxane (MAO) the palladium complexes show high activity up to 5.9 x 10(6) g(PNB) mol(Pd)(-1) h(-1) at 20 degrees C, while activities of the nickel systems are much lower, but strongly solvent dependent. Detailed studies on the dependence of activity on polymerization conditions such as molar ratios of catalyst and cocatalyst, temperature, reaction time and solvent were carried out. All obtained polynorbornenes (PNB) were noncrystalline and insoluble, but have high glass transition temperatures (T(g)). Microstructures were analyzed by IR spectroscopy and solid state (CP/MAS) (13)C NMR, revealing distinct patterns for the PNB produced by Ni- or Pd-catalysts. Structure/activity correlations deduced for the complexes with different ligand systems suggest that activities and polymer microstructures depend rather on the metal type than on ligand intricacies.

The crystal structure of 1,3,5-tris(2-fluoro-2-methylpropionylamino)benzene was solved by combining powder X-ray diffraction, solid-state NMR spectroscopy and quantum chemical calculations.

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