The structure and protein binding of amyloid-specific dye reagents.

Abstract

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

“…In biological models, however, complex dose response relationships similar to those found here were observed [17]. While it has been shown that Congo red and other aromatic heterocycles capable of interacting with proteins in cross-β-sheet conformation form disordered micelles at millimolar concentrations in aqueous solution [60,61], these compounds also form ordered aggregates at far lower, pharmacologically relevant concentrations. For example, the K dim for Congo red has been estimated as ~20 μM in water [62].…”

Research Article: The Use of Rigid, Fibrillar Congo Red Nanostructures for Scaffolding Protein Assemblies and Inducing the Formation of Amyloid‐like Arrangement of Molecules

“…Poly‐ l ‐lysine hydrobromide (molecular weight: 30 000–70 000) and Thioflavin T were purchased from Sigma‐Aldrich Co (St. Louis, MO, USA). Dye Congo red (97% purity) was from Aldrich Chemical Co (Milwaukee, WI, USA), 4,4′‐bis (1‐amino‐6‐sulphonaphtyl‐4‐azo)biphenyl was synthesized as described earlier (29). Immunoglobulin L‐chain lambda was obtained from the urine of a myeloma patient, purified by salting out followed by Sephacryl S‐300 gel filtration.…”

“…The large interaction surface ensured by the ribbon‐like supramolecular form of the dye ligands appears to be a significant factor for the strong complexation observed. Their plasticity facilitates considerably the fitting in with polypeptides rendered available at the site of dye‐binding protein (21–31). At a low pH, however, the property of the dye nanostructures changes dramatically.…”

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

“…To verify that silver added to dyes forms part of the complex, the stain and free silver ions were separated by agarose electrophoresis using 0.05 M Tris/HNO 3 buffer (pH=8.2) in order to verify that the silver ions migrate to the anode (together with dyes) and not to the cathode (as free ions). To localize the silver in the plate, agarose plates are placed in a solution of sodium dithionite to discolor CR spots and simultaneously reduce silver ions (Stopa et al 2003). Reduction continues until CR spots disappear.…”

“…Models of aggregates. Hypothetical models of aggregates were constructed on the basis of former studies regarding CR location in the V domains of immunoglobulin light chains (Stopa et al, 2003), as well as current experimental results.…”