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Self-assembly of amyloid polypeptides (1) imparts biological effects depending on the size in over 20 amyloid diseases and (2) produces useful yet relatively untapped biomaterials. Unfortunately, our understanding of amyloid polypeptides, as related to biomedical implications and biomaterial applications, is limited by their self-assembling nature. In this study, we report the creation of a dual peptide system, where a pair of β-amyloid (Aβ) variants are not self-assembled but hetero-assembled in the presence of their assembly partners. We provide evidence that the resulting hetero-assemblies share molecular, structural and morphological similarities with typical amyloid self-assemblies formed by a single polypeptide (e.g., Aβ). We anticipate that our dual peptide system may readily be adapted for precise control of amyloid assembly, for the study of size-dependent neurotoxicity and precise fabrication of amyloid biomaterials.

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Aggregations of β-amyloid (Aβ) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aβ and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aβ and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aβ fibrillization while promoting oligomerization of Aβ monomers and stabilizing preformed Aβ oligomers via co-assembly, as judged by Thioflavin T fluorescence, transmission electron microscopy and SDS-and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aβ species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the Cterminus of Aβ, when judged by competitive immunoassays using antibodies recognizing various fragments of Aβ. We also show that the C-terminus of Aβ is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aβ, and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

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A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin-binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted β-lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution.