Issue 4, 2011

Spectroscopic characterization of diverse amyloid fibrilsin vitro by the fluorescent dyeNile red

Abstract

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting Aβ1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanolwater mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of Aβ1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.

Graphical abstract: Spectroscopic characterization of diverse amyloid fibrilsin vitro by the fluorescent dye Nile red

Article information

Article type
Paper
Submitted
17 Oct 2010
Accepted
07 Jan 2011
First published
31 Jan 2011

Mol. BioSyst., 2011,7, 1232-1240

Spectroscopic characterization of diverse amyloid fibrilsin vitro by the fluorescent dye Nile red

R. Mishra, D. Sjölander and P. Hammarström, Mol. BioSyst., 2011, 7, 1232 DOI: 10.1039/C0MB00236D

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