Fluorescence correlation spectroscopy reveals a cooperative unfolding of monomeric amyloid-β 42 with a low Gibbs free energy

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.

Compact fibril-like structure of amyloid β-peptide (1–42) monomers

“…0.9-1.8 nm. [13][14][15][16][17] The average end to end distance follows a similar trend as the radius of gyration, with a value of 3.2 nm for Amber99SB*-ILDN and 2.7 and 2.8 nm for Charmm36mW without and with NaCl, respectively. The wide distributions of the end to end distance, shown in Figure S3, and the standard deviations of ∼1 nm indicate similar types of structural fluctuations in all three simulations.…”

“…10,11 In terms of overall dimensions, the hydrodynamic radius of Aβ40 was measured to be ∼1.6 nm at room temperature 12 while for Aβ42 it adopts a range of values between 0.9-1.8 nm. [13][14][15][16][17] Computationally, the Aβ42 monomer structure has been studied with various simulating techniques and force fields.…”

Imaging therapeutic peptide transport across intestinal barriers

“…161 Similarly, FCS may reveal conformation changes of fluorescently labeled peptides in cases where these changes are associated with an altered diffusion coefficient of the peptides. 162 Fluorescent labeling may also provide the opportunity of studying peptides using Förster/fluorescence resonance energy transfer (FRET). 163 This non-radiant energy transfer process may take place between two fluorophores when the emission wavelength of the one fluorophore (the donor) overlaps with the excitation wavelength of the other fluorophore (the acceptor).…”

“… 161 Similarly, FCS may reveal conformation changes of fluorescently labeled peptides in cases where these changes are associated with an altered diffusivity of the peptides. 162 …”

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

“…Aβ and polyQ have been classically and traditionally analyzed using FCS [ 47 , 61 , 62 , 63 , 64 , 65 ]. Although these aggregation-prone proteins were initially regarded as one of the high molecular weight sample for FCS measurement, FCS gradually became used for the analysis of the aggregation process and determining the aggregation-suppressive effect of molecular chaperones and drugs [ 45 , 47 , 66 ].…”