Yifat Miller

Zinc ions promote Alzheimer Aβ aggregation via population shift of polymorphic states

Although a key factor in Alzheimer’s disease etiology is enrichment of Zn2+ in aggregates, and there are data suggesting that zinc promotes aggregation, how Zn2+-Aβ coordination promotes aggregation is elusive. Here we probe the structures and mechanisms through which Zn2+ can affect amyloidosis. By covalently linking fragments (that have experiment-based coordinates) we observed that, in oligomeric Zn2+-Aβ42, Zn2+ can simultaneously coordinate intra- and intermolecularly, bridging two peptides. Zinc coordination significantly decreases the solvation energy for large Zn2+-Aβ42 oligomers and thus enhances their aggregation tendency. Zn2+ binding does not change the β-sheet association around the C-terminal hydrophobic region; however, it shifts the relative population of the preexisting amyloid polymorphic ensembles. As a result, although a parallel β-sheet arrangement is still preferred, antiparallel and other less structured assemblies are stabilized, also becoming major species. Overall, Zn2+ coordination promotes Aβ42 aggregation leading to less uniform structures. Our replica exchange molecular dynamics simulations further reproduced an experimental observation that the increasing Zn2+ concentration could slow down the aggregation rate, even though the aggregation rates are still much higher than in Zn2+-free solution.

Molecular-Level Examination of Cu2+ Binding Structure for Amyloid Fibrils of 40-Residue Alzheimer's β by Solid-State NMR Spectroscopy

Cu(2+) binding to Alzheimers β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimers senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.

Polymorphism of Alzheimer's Aβ17-42 (p3) Oligomers: The Importance of the Turn Location and Its Conformation

Abeta(17-42) (so-called p3) amyloid is detected in vivo in the brains of individuals with Alzheimers disease or Downs syndrome. We investigated the polymorphism of Abeta(17-42) oligomers based on experimental data from steady-state NMR measurements, electron microscopy, two-dimensional hydrogen exchange, and mutational studies, using all-atom molecular-dynamics simulation with explicit solvent. We assessed the structural stability and the populations. Our results suggest that conformational differences in the U-turn of Abeta(17-42) lead to polymorphism in beta-sheet registration and retention of an ordered beta-strand organization at the termini. Further, although the parallel Abeta(17-42) oligomer organization is the most stable of the conformers investigated here, different antiparallel Abeta(17-42) organizations are also stable and compete with the parallel architectures, presenting a polymorphic population. In this study we propose that 1), the U-turn conformation is the primary factor leading to polymorphism in the assembly of Abeta(17-42) oligomers, and is also coupled to oligomer growth; and 2), both parallel Abeta(17-42) oligomers and an assembly of Abeta(17-42) oligomers that includes both parallel and antiparallel organizations contribute to amyloid fibril formation. Finally, since a U-turn motif generally appears in amyloids formed by full proteins or long fragments, and since to date these have been shown to exist only in parallel architectures, our results apply to a broad range of oligomers and fibrils.

Hollow core of Alzheimer’s Aβ42 amyloid observed by cryoEM is relevant at physiological pH

Recent cryoEM density maps of Aβ42 fibrils obtained at low pH revealed two protofilaments winding around a hollow core raising the question if such tubular structures also exist at physiological pH. Based on the cryoEM measurements and on NMR data, we probe amyloid fibril organizations corresponding to the observed cryoEM density map. Our study demonstrates that the tubular Aβ42 fibril models exist at both acidic and physiological pH; however, the relative populations of the polymorphic models shift with pH. At acidic pH, the hollow core model exhibits higher population than the other models; at physiological pH, although it is less populated compared to the other models, structurally, it is stable and represents 8% of the population. We observe that only models with C termini facing the external surface of the fibril retain the hollow core under acidic and physiological conditions with dimensions similar to those observed by cryoEM; on the other hand, the hydrophobic effect shrinks the tubular cavity in the alternative organization. The existence of the hollow core fibril at physiological pH emphasizes the need to examine toxic effects of minor oligomeric species with unique organizations.

Synergistic interactions between repeats in tau protein and Aβ amyloids may be responsible for accelerated aggregation via polymorphic states.

Amyloid plaques and neurofibrillary tangles simultaneously accumulate in Alzheimer’s disease (AD). It is known that Aβ and tau exist together in the mitochondria; however, the interactions between Aβ oligomers and tau are controversial. Moreover, it is still unclear which specific domains in the tau protein can interact with Aβ oligomers and what could be the effect of these interactions. Herein, we examine three different Aβ–tau oligomeric complexes. These complexes present interactions of Aβ with three domains in the tau protein; all contain high β-structure propensity in their R2, R3, and R4 repeats. Our results show that, among these, Aβ oligomers are likely to interact with the R2 domain to form a stable complex with better alignment in the turn region and the β-structure domain. We therefore propose that the R2 domain can interact with soluble Aβ oligomers and consequently promote aggregation. EM and AFM images and dimensions revealed highly polymorphic tau aggregates. We suggest that the polymorphic tau and Aβ–tau aggregates may be largely due to repeat sequences which are prone to variable turn locations along the tau repeats.

The unique Alzheimer's β-amyloid triangular fibril has a cavity along the fibril axis under physiological conditions.

Elucidating the structure of Aβ(1-40) fibrils is of interest in Alzheimers disease research because it is required for designing therapeutics that target Aβ(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across β-strands and across β-sheets in Aβ fibrils. Experimentally, data for the three-fold symmetry structure of the Aβ(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-β units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Aβ(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed ∼5-7 Å M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 Å. Consequently, we revealed that the unique Aβ(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Aβ(1-42) fibrils, point to the relevance of a cavity in Aβ(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity.

Vibrational spectroscopy of (SO42−)∙(H2O)n clusters, n=1–5: Harmonic and anharmonic calculations and experiment

The vibrational spectroscopy of (SO4(2-)).(H2O)n is studied by theoretical calculations for n=1-5, and the results are compared with experiments for n=3-5. The calculations use both ab initio MP2 and DFT/B3LYP potential energy surfaces. Both harmonic and anharmonic calculations are reported, the latter with the CC-VSCF method. The main findings are the following: (1) With one exception (H2O bending mode), the anharmonicity of the observed transitions, all in the experimental window of 540-1850 cm(-1), is negligible. The computed anharmonic coupling suggests that intramolecular vibrational redistribution does not play any role for the observed linewidths. (2) Comparison with experiment at the harmonic level of computed fundamental frequencies indicates that MP2 is significantly more accurate than DFT/B3LYP for these systems. (3) Strong anharmonic effects are, however, calculated for numerous transitions of these systems, which are outside the present observation window. These include fundamentals as well as combination modes. (4) Combination modes for the n=1 and n=2 clusters are computed. Several relatively strong combination transitions are predicted. These show strong anharmonic effects. (5) An interesting effect of the zero point energy (ZPE) on structure is found for (SO4(2-)).(H2O)(5): The global minimum of the potential energy corresponds to a C(s) structure, but with incorporation of ZPE the lowest energy structure is C2v, in accordance with experiment. (6) No stable structures were found for (OH-).(HSO4-).(H2O)n, for n<or=5.

Interactions between Aβ and Mutated Tau Lead to Polymorphism and Induce Aggregation of Aβ-Mutated Tau Oligomeric Complexes

One of the main hallmarks of the fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) is the accumulation of neurofibrillary tangles in the brain as an outcome of the aggregation of mutated tau protein. This process occurs due to a number of genetic mutations in the MAPT gene. One of these mutations is the ∆K280 mutation in the tau R2 repeat domain, which promotes the aggregation vis-à-vis that for the wild-type tau. Experimental studies have shown that in Alzheimer’s disease Aβ peptide forms aggregates both with itself and with wild-type tau. By analogy, in FTDP-17, it is likely that there are interactions between Aβ and mutated tau, but the molecular mechanisms underlying such interactions remain to be elucidated. Thus, to investigate the interactions between Aβ and mutated tau, we constructed fourteen ∆K280 mutated tau-Aβ17-42 oligomeric complexes. In seven of the mutated tau-Aβ17-42 oligoemric complexes the mutated tau oligomers exhibited hydrophobic interactions in their core domain, and in the other seven mutated tau-Aβ17-42 oligoemric complexes the mutated tau oligomers exhibited salt-bridge interactions in their core domain. We considered two types of interactions between mutated tau oligomers and Aβ oligomers: interactions of one monomer of the Aβ oligomer with one monomer of the mutated tau oligomer to form a single-layer conformation, and interactions of the entire Aβ oligomer with the entire mutated tau oligomer to form a double-layer conformation. We also considered parallel arrangements of Aβ trimers alternating with mutated tau trimers in a single-layer conformation. Our results demonstrate that in the interactions of Aβ and mutated tau oligomers, polymorphic mutated tau-Aβ17-42 oligomeric complexes were observed, with a slight preference for the double-layer conformation. Aβ trimers alternating with mutated tau trimers constituted a structurally stable confined β-structure, albeit one that was energetically less stable than all the other constructed models.

Effects of mutations in de novo designed synthetic amphiphilic β-sheet peptides on self-assembly of fibrils

The self-assembly of two similar amphiphilic peptides into fibril structures is described. Molecular dynamic simulations show that both can organize similarly in a monolayer, but in the fibril bilayer, one prefers a single organization while the other forms two conformational variants. This assembly difference correlates well with our experimental results.

Ultrafast phase transitions in metastable water near liquid interfaces

Electron spectroscopy for chemical analysis (ESCA) is a powerful tool for the quantitative analysis of the composition and the chemical environment of molecular systems. Due to the lack of compatibility of liquids and vacuum, liquid-phase ESCA is much less well established. The chemical shift in the static ESCA approach is a particularly powerful observable quantity for probing electron orbital energies in molecules in different molecular environments. Employing high harmonics of 800 nm (40 eV), near-infrared femtosecond pulses, and liquid-water microbeams in vacuum we were able to add the dimension of time to the liquid interface ESCA technique. Tracing time-dependent chemical shifts and energies of valence electrons in liquid interfacial water in time, we have investigated the timescale and molecular signatures of laser-induced liquid-gas phase transitions on a picosecond timescale.