On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy.

Jelica Milošević,Natalija Polović,Radivoje Prodanović

doi:10.3390/molecules26040970

Abstract

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

Highlights

Amyloid fibrils are highly ordered protein aggregates with monotonous cross-β structure independent of the sequence [1]
A certain group of authors takes some forms of protofibrils as oligomeric intermediates, while others reserve this term for spherical intermediates that precede fibrillar forms
The analysis and detection of oligomeric amyloid intermediates are of crucial importance as these are the cytotoxic agent on the road to amyloid fibrils

Summary

Introduction

Amyloid fibrils are highly ordered protein aggregates with monotonous cross-β structure independent of the sequence [1]. Lag stage of amyloid misfolding, the native state of a protein is destabilized and transformed into nuclei for fibrillation Once formed they can proceed to metastable oligomers which are considered the actual toxic form inducing misfolding-related pathological states [3]. Oligomer is used as a term for a state that excludes 50% of protein molecules in a monomer state and the other case where more than 50% is part of a big cluster [4]. They continue their route to fibrils by occupying protofibril form—wormlike filamentous late intermediate stage without periodicity in their structure [5,6]. Protofibrils assemble into protofilaments which represent single-stranded fibrils that twist helically to form mature fibrils

Methods

Results

Conclusion