Abstract
The amyloid-beta peptide (Aβ) plays a major role in the progression of Alzheimer’s disease. Due to its high toxicity, the 42 amino acid long isoform Aβ42 has become of considerable interest. The Aβ42 monomer is prone to aggregation down to the nanomolar range which makes conventional structural methods such as NMR or X-ray crystallography infeasible. Conformational information, however, will be helpful to understand the different aggregation pathways reported in the literature and will allow to identify potential conditions that favour aggregation-incompetent conformations. In this study, we applied fluorescence correlation spectroscopy (FCS) to investigate the unfolding of Alexa Fluor 488 labelled monomeric Aβ42 using guanidine hydrochloride as a denaturant. We show that our Aβ42 pre-treatment and the low-nanomolar concentrations, typically used for FCS measurements, strongly favour the presence of monomers. Our results reveal that there is an unfolding/folding behaviour of monomeric Aβ42. The existence of a cooperative unfolding curve suggests the presence of structural elements with a Gibbs free energy of unfolding of about 2.8 kcal/mol.
Highlights
Aggregation of amyloid-beta (Aβ) is believed to be one of the key processes in the development and progression of Alzheimer’s disease (AD)
amyloid-β 42 (Aβ42) was prepared from recombinant C(0)Aβ42 after Alexa Fluor 488 (AF488) coupling to the cysteine residue and subsequent size exclusion chromatography (SEC) (Fig. S1 in the Supplementary Information)
In order to be sure that the unfolding transition is solely due to conformational changes of the monomers, we performed Photon counting histogram (PCH) analysis on the data set of the fluorescence correlation spectroscopy (FCS) unfolding experiments, at the lowest GdnHCl concentration (0.25 M) and at a high GdnHCl concentration (5 M)
Summary
Aggregation of amyloid-beta (Aβ) is believed to be one of the key processes in the development and progression of Alzheimer’s disease (AD). Information about Aβ42 monomer conformation exists so far only from solutions containing hexafluoroisopropanol (HFIP)[1, 2]. These conditions are far from being physiological, especially because typical Aβ42 concentrations in vivo are in the nanomolar range[3]. It is suggested that the Aβ42 monomer might exist in equilibrium between a folded and unfolded conformer[4], but to date there is no substantial experimental evidence for this hypothesis. Since FCS measurements are typically performed at low nanomolar concentrations, this technique is well-suited to study dye-labelled Aβ42 monomers. The Gibbs free energy of unfolding was determined to be about 2.8 kcal/
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