Moderate heating (40–50°C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon‐like micellar bodies. The L chain λ dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye–protein (L λ chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40–45°C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55°C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL‐VL domain incoherence, and protein melting. Of these three possibilities, local low‐energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N‐terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19‐amino acid N‐terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye–protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74–80 and 105–110, as shown by model analysis. The character of the temperature‐induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 446–456, 2001

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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.

The ordered amyloid-like organization of protein aggregates was obtained using for their formation the rigid fibrillar nanostructures of Congo red as the scaffolding. The higher rigidity of used dye nanoparticles resulted from the stronger stacking of molecules at low pH (near the pK of the dye amino group) because of the decreased charge repulsion. The polylysine, human globin, and immunoglobulin L chain were arranged in this way to form deposits of amyloid properties. The scaffolding was introduced simply by mixing the dye and proteins at a low pH or the dye was used in the preorganized form by maintaining it in the electric field before and during protein addition. The polarization and electron microscopy studies confirmed the unidirectional organization of the complex. The precipitate of the complex was used for studies directly or after the partial or complete removal of the dye. The results suggest that the process of formation of amyloid-like deposits may bypass the nucleation step. It is possible if the protein aggregation occurs in unidirectionally organized (because of scaffolding) assembly of molecules, arranged prior to self-association. The recognition of the structure of amphoteric Congo red nanoparticles used for the scaffolding was based on the molecular dynamics simulation.

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