Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies

J Mol Biol. 2009 Jan 30;385(4):1052-63. doi: 10.1016/j.jmb.2008.11.006. Epub 2008 Nov 14.


A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic" (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of beta-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM beta-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the beta-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more beta-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Benzothiazoles
  • Binding Sites
  • Crystallography, X-Ray
  • Models, Biological
  • Models, Molecular
  • Peptides / chemistry*
  • Peptides / metabolism*
  • Protein Structure, Secondary
  • Surface Properties
  • Thiazoles / chemistry
  • Thiazoles / metabolism*
  • Tyrosine / metabolism


  • Benzothiazoles
  • Peptides
  • Thiazoles
  • thioflavin T
  • Tyrosine

Associated data

  • PDB/3EC5