Formation of amyloid fibrils by Aβ42 protein is a pathological hallmark of Alzheimer’s disease. Aβ42 fibrillization is a nucleation-dependent polymerization process, in which nucleation is the rate-limiting step. Structural knowledge of the fibril nucleus is important to understand the molecular mechanism of Aβ aggregation and is also critical for successful modulation of the fibrillization process. Here, we used a scanning mutagenesis approach to study the role of each residue position in Aβ42 fibrillization kinetics. The side chain we used to replace the native residue is a nitroxide spin label called R1, which was introduced using site-directed spin labeling. In this systematic study, all residue positions of Aβ42 sequence were studied, and we identified six key residues for the Aβ42 fibril formation: H14, E22, D23, G33, G37, and G38. Our results suggest that charges at positions 22 and 23 and backbone flexibilities at positions 33, 37, and 38 play key roles in Aβ42 fibrillization kinetics. Our results also suggest that the formation of a β-strand at residues 15–21 is an important feature in Aβ42 fibril nucleus. In overall evaluation of all of the mutational effects on fibrillization kinetics, we found that the thioflavin T fluorescence at the aggregation plateau is a poor indicator of aggregation rates.

In yeast, the formation of Ure2 fibrils underlies the prion state [URE3], in which the yeast loses the ability to distinguish good nitrogen sources from bad ones. The Ure2 prion domain is both necessary and sufficient for the formation of amyloid fibrils. Understanding the structure of Ure2 fibrils is important for understanding the propagation not only of the [URE3] prion but also of other yeast prions whose prion domains share similar features, such as the enrichment of asparagine and glutamine residues. Here, we report a structural study of the amyloid fibrils formed by the Ure2 prion domain using sitedirected spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We completed a spin label scanning of all the residue positions between 2 and 80 of the Ure2 prion domain. The EPR data show that the Ure2 fibril core consists of residues 8−68 and adopts a parallel in-register β-sheet structure. Most of the residues show strong spin−exchange interactions, suggesting that there are only short turns and no long loops in the fibril core. Based on the strength of spin−exchange interactions, we determined the likely locations of the β-strands. EPR data also show that the C-terminal region of the Ure2 prion domain is more disordered than the N-terminal region. The roles of hydrophobic and charged residues are analyzed. Overall, the structure of Ure2 fibrils appears to involve a balance of stabilizing interactions, such as asparagine ladders, and destabilizing interactions, such as stacking of charged residues.

Deposition of Aβ42 aggregates in the form of amyloid plaques is a pathological hallmark of Alzheimer’s disease. A desired avenue of intervention is the inhibition of Aβ42 aggregation. Epigallocatechin gallate (EGCG), the main polyphenol in green tea, has been generally considered an inhibitor of Aβ aggregation. However, previous experiments focused on the reduction of the amount of Aβ42 aggregates, while the effect of EGCG on the rate of Aβ42 aggregation was not critically analyzed. Here we performed an experimental evaluation of Aβ42 aggregation kinetics in the absence and presence of EGCG at a wide range of concentrations. We found that EGCG reduced thioflavin T fluorescence in an EGCG concentration-dependent manner, suggesting that EGCG reduced the amount of Aβ42 fibrils. The effect of EGCG on the rate of Aβ42 aggregation appears to be bimodal. We found that higher EGCG-to-Aβ42 ratios promoted the rate of Aβ42 aggregation, while lower EGCG-to-Aβ42 ratios inhibited the aggregation rate. To confirm that the reduction of thioflavin T fluorescence is due to the lowered aggregate quantity, but not due to perturbation of thioflavin T binding to Aβ42 fibrils, we probed the effect of EGCG on Aβ42 aggregation using site-directed spin labeling. Electron paramagnetic resonance of spin-labeled Aβ42 aggregates suggests that high EGCG-to-Aβ42 ratios led to a greatly reduced amount of Aβ42 fibrils, and these aggregates adopt similar structures as the fibrils in the no-EGCG sample. Potential implications of this work in designing prevention or therapeutic strategies using EGCG are discussed.

Amyloid formation is involved in a wide range of neurodegenerative diseases including Alzheimer's and prion diseases. Structural understanding of the amyloid is critical to delineate the mechanism of aggregation and its pathological spreading. Site-directed spin labelling has emerged as a powerful structural tool in the studies of amyloid structures and provided structural evidence for the parallel in-register β-sheet structure for a wide range of amyloid proteins. It is generally accepted that spin labelling does not disrupt the structure of the amyloid fibrils, the end product of protein aggregation. The effect on the rate of protein aggregation, however, has not been well characterized. Here, we employed a scanning mutagenesis approach to study the effect of spin labelling on the aggregation rate of 79 spin-labelled variants of the Ure2 prion domain. The aggregation of Ure2 protein is the basis of yeast prion [URE3]. We found that all spin-labelled Ure2 mutants aggregated within the experimental timeframe of 15 to 40 h. Among the 79 spin-labelled positions, only five residue sites (N23, N27, S33, I35 and G42) showed a dramatic delay in the aggregation rate as a result of spin labelling. These positions may be important for fibril nucleation, a rate-limiting step in aggregation. Importantly, spin labelling at most of the sites had a muted effect on Ure2 aggregation kinetics, showing a general tolerance of spin labelling in protein aggregation studies.