Silver ions as EM marker of congo red ligation sites in amyloids and amyloid-like aggregates

Abstract

Congo red (CR) which is known to act as selective amyloid ligand may when binds to these protein forms reflect their internal molecular structure. The disclosure by EM of sites binding the dye and their distribution in amyloids and amyloid-like aggregates formed in vitro is the focus of our work. In order to produce the required contrast, CR has been indirectly combined with metal via including by intercalation of Titan yellow (TY) which exhibits relatively strong affinity for silver ions. The resulting combined ligand retains its ability to bind to proteins which it owes to CR and can easily be detected in EM studies thanks to TY less active in penetration to proteins. We have found however that in protein aggregates where unfolding is stabilized by aggregation and therefore irreversible, TY alone may serve as both ligand and metal carrier.

The formation of ordered structures in amyloids were studied using IgG light chains with amyloidogenic properties, converted into amyloids through shaking. The resulting EM images were subjected to interpretation on the basis of the authors’ earlier research into the CR/light chain complexation process. Results indicate that dimeric light chains, which are the subject of our study, produce amyloids or amyloid-like complexes with chain-like properties and strong helicalization tendencies. Cursory analysis suggests that edge polypeptide loops belonging to unstable light chains form intermolecular bridges which promote creation of loose gel deposits, or are otherwise engaged in swapping processes leading to higher structural ordering.