Dataset Information

Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence.

ABSTRACT: Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-beta peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of A beta(1-40.) Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the A beta(1-40) peptide forms a weak complex with neuroserpin (dissociation constant of 10 +/- 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The A beta(1-40) is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.

SUBMITTER: Chiou A

PROVIDER: S-EPMC2764104 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence.

Chiou Albert A Hägglöf Peter P Orte Angel A Chen Allen Yuyin AY Dunne Paul D PD Belorgey Didier D Karlsson-Li Susanna S Lomas David A DA Klenerman David D

Biophysical journal 20091001 8

Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-beta peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluor ...[more]

PMID: 19843463

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Project description:A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

Dataset Information

Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence.

Publications

Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence.

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