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Identifying Insulin Fibril Conformational Differences by Thioflavin-T Binding Characteristics

Mantas Ziaunys1, Andrius Sakalauskas1, Vytautas Smirnovas1

  • 1Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio al. 7, Vilnius LT-10257, Lithuania.

Biomacromolecules
|November 17, 2020
PubMed

Insights

This study reveals that the fluorescent probe thioflavin-T (ThT) exhibits varying fluorescence properties and binding behaviors depending on the specific conformation of insulin amyloid fibrils, impacting its use in tracking fibril formation.

Area of Science:

  • Biochemistry
  • Biophysics
  • Materials Science

Background:

  • Amyloidogenic protein aggregation causes over 30 amyloidoses, including neurodegenerative diseases.
  • Current treatments for amyloidosis remain limited, necessitating further research into disease mechanisms.
  • Thioflavin-T (ThT) is widely used to monitor amyloid formation, but its interactions with fibrils are not fully understood.

Purpose of the Study:

  • To investigate the influence of different insulin fibril conformations on ThT binding and fluorescence.
  • To characterize distinct insulin fibril structures using multiple analytical techniques.
  • To assess the reliability of ThT as a universal probe for amyloid fibrils.

Main Methods:

  • Generation of insulin fibrils under four distinct conditions.
  • Characterization using atomic force microscopy (AFM) and Fourier-transform infrared spectroscopy (FTIR).
  • Analysis of ThT binding affinity and fluorescence quantum yield for different fibril conformations.

Main Results:

  • Significant variations in ThT fluorescence quantum yields were observed across different insulin fibril conformations.
  • Excitation/emission maxima positions of ThT differed based on fibril structure.
  • Distinct ThT binding modes were identified for the various insulin fibril conformations.

Conclusions:

  • Insulin fibril conformation significantly impacts ThT probe behavior.
  • The findings highlight the need to consider fibril conformation when interpreting ThT fluorescence data.
  • This research provides crucial insights into the limitations and nuances of using ThT in amyloid studies.

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