A Palette of Fluorescent Aβ42 Peptides Labelled at a Range of Surface-Exposed Sites

Abstract

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of Aβ42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled Aβ42 peptides that can be used to gain understanding of the complex mechanisms of Aβ42 self-assembly and help to develop a more targeted approach to cure the disease.

Design of amyloidogenic peptide traps

“…To track the degree of monomer conversion into fibrils, ThT was added exclusively to control wells, and the fibrils were harvested from ThT-free sample wells after the plateau was reached in the control wells.To perform binding experiments of monomeric Aβ with the binders, cysteine-carrying Aβ mutant (S8C) has been expressed and purified as described previously (Thacker et al 2022). Briefly, the plasmid carrying synthetic genes with E. coli optimized codons for S8C mutant (developed by Thacker and colleagues and purchased from Genscript) were transformed into BL21 DE3 pLysS star E. coli strain and the protein was expressed in auto-induction medium (Studier 2005).…”

Ganglioside Micelles Affect Amyloid β Aggregation by Coassembly

“…A cysteine mutant (S8C) of Aβ42 was covalently labeled with Alexa-647 (Alexa-Aβ42) using maleimide chemistry. Position 8 was chosen for labeling because it has been shown to give similar aggregation kinetics and fibril morphology as wild-type Aβ42 . The labeled protein (20 nM) was mixed with varying amounts of unlabeled GM1 (12–1000 μM) above the CMC of GM1 (Figure S5).…”

“…Position 8 was chosen for labeling because it has been shown to give similar aggregation kinetics and fibril morphology as wild-type Aβ42. 45 The labeled protein (20 nM) was mixed with varying amounts of unlabeled GM1 (12−1000 μM) above the CMC of GM1 (Figure S5). The Aβ42 concentration was chosen to not exceed the reported Aβ42 solubility (∼20 nM), 46 meaning that Aβ42 self-aggregation at this concentration can be neglected, at least for the time frame of the experiment (5 min).…”

“…The Aβ42 or Aβ40 monomers with and without GM1 were incubated at 37 °C in PEGylated polystyrene plates (Corning 3881) in a plate reader for several days before they were collected for cryo-TEM experiments. The preparation of samples for cryo-TEM was performed as previously reported . The specimens were collected and plunged using an automatic plunge freezer system (Leica EM GP), which is set at a controlled chamber temperature and relative humidity.…”

An aggregation inhibitor specific to oligomeric intermediates of Aβ42 derived from phage display libraries of stable, small proteins

“…Aβ(M1-40) with the sequence MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, here called Aβ40, and Aβ(M1-42) with the sequence MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, here called Aβ42, were recombinantly expressed in E. coli from PetSac plasmids containing synthetic genes with E. coli optimized codons and purified from inclusion bodies using sonication, ion exchange, and two rounds of size exclusion chromatography, and the isolated monomers were stored as lyophilized aliquots as previously described ( 8 , 14 , 48 ). Aβ42-S8C was expressed and purified in the same way except that 1 mM DTT was included in all buffers up to the last SEC step, which served to isolate monomers in 20 mM phosphate buffer, pH 8.0, and to remove excess DTT ( 49 ).…”