How Fluorescent Tags Modify Oligomer Size Distributions of the Alzheimer Peptide

Abstract

Within the complex aggregation process of amyloidogenic peptides into fibrils, early stages of aggregation play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low-molecularweight aggregates of the Alzheimer amyloid-b peptide (Ab) have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation-related diseases. One of the main techniques used to characterize oligomeric stages is fluorescence spectroscopy. To this end, Ab peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. In this investigation, we systematically study the effects of four of the most commonly used fluorophores on the aggregation of Ab (1-40). Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, and transmission electron microscopy, atomic force microscopy and x-ray diffraction to investigate the structure of mature fibrils. Although the structures of the fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high-molecular-weight oligomers of Ab (1-40) (as found for the fluorophores HiLyte 647 and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488 and Atto 488. The latter leads for Ab (1-40) to low-molecular-weight oligomers only, which is in contrast to Ab (1-42). The disease-relevant peptide Ab (1-42) displays high-molecular-weight oligomers even in the absence of significant attractive fluorophore-peptide interactions. Hence, our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates, which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.

Fluorescence Phenomena in Amyloid and Amyloidogenic Bionanostructures

“…The early state of Aβ(1-40) fibrillation was also explored using time-resolved and single-molecule FL experiments by Ott's research group [72]. In their studies, the authors conjugated several fluorophores like Atto 488, Atto 655, Hi-Lyte 488 and HiLyte 647 on the polypeptide sequence.…”

An α-Cyanostilbene Derivative for the Enhanced Detection and Imaging of Amyloid Fibril Aggregates

“…Thus, singlemolecule approaches that enable the characterisation of individual amyloid fibrils are becoming increasingly common [14][15][16][17][18]61,70 . The use of AIE fluorophores has a substantial advantage in singlemolecule investigations of amyloid formation since photobleaching is avoided and the protein of interest does not need to be covalently labelled with a fluorescent probe, which can change the kinetics and overall morphology of the elongating fibril 17,61,[70][71][72][73][74] . With this in mind, we sought to investigate if ASCP could serve as a suitable alternative to ThT in single-molecule TIRF microscopy experiments.…”

Visualizing and trapping transient oligomers in amyloid assembly pathways

“…An additional factor which must be considered is that most single particle fluorescence experiments make use of fluorescently-labelled protein samples, and therefore rely on the assumption that the incorporation of fluorescent dyes, which are often large and hydrophobic, does not perturb the assembly mechanism. This assumption may not always hold true [ [84] , [85] , [86] ], and the appropriate selection of fluorophores must be ensured by performing thorough control experiments. It is also possible to perform experiments with fluorescent probes which interact non-covalently with the protein of interest, and which can therefore be added after assembly has taken place [ 81 , 82 , 87 , 88 ].…”