NMR Reveals Two-Step Association of Congo Red to Amyloid β in Low-Molecular-Weight Aggregates

Aggregation of the Amyloid beta peptide into amyloid fibrils is closely related to development of Alzheimer s disease Many small aromatic compounds have been found to act as inhibitors of fibril formation, and have inspired the search for new drug candidates However, the detailed mechanisms of inhibition are largely unknown In this study, we have examined in detail the binding of the fibril-formation inhibitor Congo Red (CR) to monomeric A beta(1-40) using a combination of 1D 2D, saturation transfer difference, and diffusion NMR as well as dynamic light scattering experiments Our results show that CR binds to the fibril forming stretches of A(beta 1-40) monomers and that complex formation occurs in two steps An initial 1 1 CR A beta(1-40) complex is formed by a relatively strong interaction (K-d approximate to 5 mu M) and a 2 1 complex is formed by binding another CR molecule in a subsequent weaker binding step (K-d approximate to 300 mu M) The size of these complexes is comparable to that of A beta(1-40) alone The existence of two different complexes might explain the contradictory reports regarding the inhibitory effects of CR on the fibril-formation process