Dataset Information

Amyloid ? Oligomeric Species Present in the Lag Phase of Amyloid Formation.

ABSTRACT: Alzheimer's disease (AD)-associated amyloid ? peptide (A?) is one of the main actors in AD pathogenesis. A? is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on A?42 and its oligomeric assemblies in the lag phase of amyloid formation, as studied by sedimentation velocity (SV) centrifugation. The assembly state of A? during the lag phase, the time required by an A? solution to reach the exponential growth phase of aggregation, was characterized by a dominant monomer fraction below 1 S and a population of oligomeric species between 4 and 16 S. From the oligomer population, two major species close to a 12-mer and an 18-mer with a globular shape were identified. The recurrence of these two species at different initial concentrations and experimental conditions as the smallest assemblies present in solution supports the existence of distinct, energetically favored assemblies in solution. The sizes of the two species suggest an A?42 aggregation pathway that is based on a basic hexameric building block. The study demonstrates the potential of SV analysis for the evaluation of protein aggregation pathways.

SUBMITTER: Wolff M

PROVIDER: S-EPMC4449029 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation.

Wolff Martin M Unuchek Dmitry D Zhang Bo B Gordeliy Valentin V Willbold Dieter D Nagel-Steger Luitgard L

PloS one 20150529 5

Alzheimer's disease (AD)-associated amyloid β peptide (Aβ) is one of the main actors in AD pathogenesis. Aβ is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on Aβ42 and its oligomeric ...[more]

PMID: 26024352

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Project description:Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (F(C[triple bond]N)) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by F(C[triple bond]N). The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. F(C[triple bond]N) fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and F(C[triple bond]N) fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until beta-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. F(C[triple bond]N) also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. F(C[triple bond]N) is readily accommodated into proteins; thus, the approach should be broadly applicable.

Dataset Information

Amyloid ? Oligomeric Species Present in the Lag Phase of Amyloid Formation.

Publications

Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation.

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