Fluorescence-Labeled Amyloid Beta Monomer: A Molecular Dynamical Study

Abstract

The aggregation process of the Amyloidβ (Aβ) peptide is one of the central questions in Alzheimers’s research. Fluorescence-labeled single-molecule detection is a novel technique concerning the early stage investigation of Aβ aggregation, where the labeling dyes are covalently bound to the Aβ monomer. As the influence of the dye on the conformational space of the Aβ monomer can be significant, its effect on the seeding process is an open question. The applied fluorescent molecule continuously switches between an active (ON) and an inactive (OFF) state, where the latter supports an extra rotational restriction at many commercially available dyes. However, only a few theoretical studies simulated the Aβ monomer in the presence of a dye and none of them considered the difference between the ON and the OFF states. Therefore, we examined the impact of a selected fluorescence dye (Alexa 568) on the conformational space of the monomeric Aβ(1–42) peptide in its ON and OFF state by replica exchange molecular dynamic simulations. Investigations on secondary structure elements as well as dye-peptide contact analysis for the monomers are presented. Experimental and theoretical NMR shifts were contrasted to qualify the calculation protocol and theoretical values of the labeled and the non-labeled peptide were also compared. We found that the first five residues have higher helical propensity in the presence of the dye, and electrostatic properties could strongly affect the connection between the dye and the peptide parts.

A robust yet simple method to generate fluorescent amyloid nanofibers

“…[4][5][6] Studies that use fluorophore-tagged proteins greatly advance the mechanistic understanding of the cellular internalization of protein amyloids and their potential to intervene in cellular metabolism. 4,5,7,8 Usually, fluorescent proteins are synthesized by tagging the desired fluorophore to the peptide backbone or side chains at an appropriate position through a convenient covalent bond. 6,7,9,10 For example, thiol groupmediated cross-linking is performed using cysteine amino acids present in the peptide sequence.…”

“…Importantly, the presence of the fluorophore-molecule, as an inbuilt moiety in the polypeptide chain, may influence the inherent native conformation of the proteins, which could possibly affect both the aggregation propensity and the nanoarchitecture of their amyloid structures. [8][9][10] Due to the abovementioned complications associated with the covalent tagging of fluorophores, it is important for the development of affordable and simpler methods for making fluorescent amyloid nanostructures.…”

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-β Ligand Binding

“…The contrast observed between the two flexible termini correlates with their possible involvement in oligomerization dynamics. 63 We note that labeling may alter the intrinsic properties of biomolecules of interest, 64,65 however, there were only insignificant differences in the conformational ensembles of unlabeled monomeric Aβ and each labeling isomer (Supporting Information Figures S9− S10), as well as identical copper ion binding sites and similar affinities (Supporting Information Figure S3).…”

An efficient method to generate fluorescent amyloid fibrils

“…[1–3] Hence, it becomes important to understand various biophysical properties of the amyloids and their toxic effects on vital cellular activities. [4–6] Use of fluorophore-tagged peptides and proteins has been facilitating the mechanistic understanding of the internalization and the localisation of amyloid aggregates and their intervention with crucial cellular metabolism as well [4, 5, 7, 8]. Usually, a fluorescent amyloidogenic peptide is synthesized by tagging the desired fluorophore at an appropriate position in the peptide backbone or side chains through a convenient covalent bond [6, 7, 9, 10].…”

“…Though use of fluorophore-tagged peptides has become a vital tool for amyloid research, this protocol is not free from complications including its high cost and the difficulties in both the synthesis and the yield of the desired peptides. More importantly, the presence of the fluorophore molecule, as an inbuilt moiety in the polypeptide chain, may influence the inherent properties of the proteins including their actual conformation, their aggregation propensity, and the architecture of their amyloid assembly[8–10]. Due to the existence of the above-mentioned complications with the covalent tagging of fluorophores with peptides, it is very important for development of an affordable and simpler method for making fluorescent amyloid structures.…”