Heterogeneity and Turnover of Intermediates during Amyloid‑β (Aβ) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy

Self-assembly of amyloid β (Aβ) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among Aβ molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. Aβ self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding Aβ aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying Aβ aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of Aβ peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 × 10⁶ kDa and revealed the hitherto unobserved kinetic turnover of intermediates during Aβ aggregation. The effect of different Aβ concentrations, Aβ:ThT ratios, differences between the 40 (Aβ40) and 42 (Aβ42) residue long variants of Aβ, and the effect of stirring were also examined

Ionic Strength Modulation of the Free Energy Landscape of Aβ₄₀ Peptide Fibril Formation

Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimer’s disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (F_β*) and a mature stable state (F_β), where salt favors formation of the F_β fibril morphology. Addition of salt to preformed F_β* accelerates transition to F_β, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the F_β state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states

Specific Binding of a β-Cyclodextrin Dimer to the Amyloid β Peptide Modulates the Peptide Aggregation Process

Alzheimer’s disease involves progressive neuronal loss. Linked to the disease is the amyloid β (Aβ) peptide, a 38–43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. β-Cyclodextrin consists of seven α-d-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of Aβ. We have studied the interaction between Aβ and a β-cyclodextrin dimer, consisting of two β-cyclodextrin monomers connected by a flexible linker. The β-cyclodextrin monomer has been found to interact with Aβ(1–40) at sites Y10, F19, and/or F20 with a dissociation constant (<i>K</i>_D) of 3.9 ± 2.0 mM. Here ¹H–¹⁵N and ¹H–¹³C heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the β-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the β-cyclodextrin dimer (apparent <i>K</i>_D of 1.1 ± 0.5 mM) for Aβ(1–40) compared to that of the β-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05–5 mM decreases the lag time of Aβ aggregation, while a concentration of 10 mM increases the lag time. The β-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the Aβ(1–40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by Aβ(1–40) alone are replaced by a major fraction of amorphous aggregates in the presence of the β-cyclodextrin dimer

The Neuronal Tau Protein Blocks <i>in Vitro</i> Fibrillation of the Amyloid‑β (Aβ) Peptide at the Oligomeric Stage

In Alzheimer’s disease, amyloid-β (Aβ) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aβ and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aβ₄₀ peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aβ₄₀ peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aβ₄₀ peptides. The Aβ monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aβ fibrillation

An NMR and MD Modeling Insight into Nucleation of 1,2-Alkanediols: Selective Crystallization of Lipase-Catalytically Resolved Enantiomers from the Reaction Mixtures

The work on developing a scalable lipase-catalytic method for the kinetic resolution of long-chain 1,2-alkanediols, complemented by crystallization of the pure enantiomers from the reaction mixtures, offered the possibility of a more detailed study of the aggregation of such diols. MD modeling, mass spectrometry, ¹H NMR, and DOSY studies provided a novel insight into the nucleation process. An efficient protocol for stereo- and chemoselective crystallization of (<i>S</i>)-<b>1</b>,2-dodecanediol and related compounds from the crude bioconversion mixtures was developed