Barbara Stopa

Self‐assembly of Congo Red—A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions

The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. n n n nTwo different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. n n n nMolecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr.

Supramolecular ligands: Monomer structure and protein ligation capability

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.

Congo red-stabilized intermediates in the λ light chain transition from native to molten state

Disruption of tertiary interaction makes a protein accessible to penetration by different small molecular compounds. Their interaction may stabilize the altered protein conformation. Congo red is proposed here as a stabilizer of the molten globule state and also of highly reversible intermediates in the transition from native to molten state. Human immunoglobulin lambda light chain (dimer) was used. Two protein-Congo red complexes were found after heating lambda chain in the presence of Congo red. They differed in the amount of attached dye molecules. The binding of dye was interpreted as a two-step dye penetration process involving the peripheral parts of the protein in the first step (at lower temperatures). It was concluded that the liquid crystal properties of Congo red enable it to form specific complexes with proteins, which have become accessible to penetration by ligands after global or local disruption of tertiary interaction. This dye may thus be used as a stabilizer of unfolding intermediates in the step preceding the molten globule state.

Congo Red bound to α-1-proteinase inhibitor as a model of supramolecular ligand and protein complex

The complex formation and structure of α-1-proteinase inhibitor with supramolecular ligand Congo Red was predicted using molecular mechanics and molecular dynamics simulation. A seven-molecule Congo Red ligand was introduced to the cleft in β-sheet “A” of an α-1-proteinase inhibitor in place of the peptide chain fragment (342-358) which occupies this locus in the cleaved form of the inhibitor. The striking similarity of Congo Red and peptide chain (342-358) insertion effects, observed by comparison of root mean square (r.m.s.)–distance plots as protein stability increased, confirmed the reliability of the constructed complex. The binding predicted theoretically for the one available cleft in the β-sheet, limited to a few Congo Red molecules, was verified experimentally. α-1-proteinase inhibitor was chosen for this study because of the known natural instability of its β-pleated sheet, but the model is believed to represent other Congo Red complexes involving proteins whose accessibility for dye penetration may be triggered by function-derived structural alterations or may be generated in unfolding conditions.

The conformational characteristics of Congo red, Evans blue and Trypan blue.

The structures of the closely related bis-azo dyes Evans blue, Trypan blue and Congo red, which appeared to have different self-assembly properties and correspondingly different abilities to form complexes with amyloids and some other proteins, were compared in this work. Ab initio and semi-empirical methods were used to find the optimal structures and partial charge distributions of the dyes. The optimal structures were searched using different widely used programs. The structures of Congo red and evans blue were found to be planar, except for the torsion on the central diphenyl bond connecting the two halves of the dye. Both symmetrical parts of the molecules appeared very close to planarity. However, Trypan blue exhibits non planarity on the di-azo bonds, as well as on the central bond between the symmetrical parts of the dye. In a consequence, the non planarity of this molecule is higher than in the case of its isomer, Evans blue and Congo red as well. The extra rotation around the azo bonds extorted by the close proximity of the sulfonic groups may be the direct cause of its poor self-assembling and complexation properties versus Evans blue.

Heat-induced formation of a specific binding site for self-assembled congo red in the V domain of immunoglobulin L chain ?

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.

Effect of self association of bis-ANS and bis-azo dyes on protein binding

A correlation was found between the ability of dyes (ANS, bis-ANS, Congo red, Evans blue) to form self-associated supramolecular structures in water and their tendency to form complexes with proteins. The self-association ability of dyes was measured as the resistance of a molecular sieve to their penetration. Quantitative evaluation of dye-protein interaction involved measuring the effect of dye on antibodies that agglutinate sheep red blood cells. Enhancement of agglutination by dye was assumed to represent its protein complexation ability. The results confirm that, relative to monomers, self-associated ligands also have altered protein binding properties.

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

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.

Why do Congo Red, Evans Blue, and Trypan Blue differ in their complexation properties?

Congo Red, Evans Blue, and Trypan Blue dyes were evaluated in terms of their ability to form supramolecular systems in water solution. A geometric transformation set was defined to construct a supramolecular model system composed of eight dye molecules. The stability of the constructed multimolecular systems was estimated by molecular dynamics using AMBER 4.1 and DISCOVER force fields. The results essentially confirm the tendency toward self‐assembly in each case. However, Congo Red and Evans Blue were found to form stable, continuous, ribbon‐like supramolecular organizations, whereas Trypan Blue self‐assembly appeared defective; some additional deviations from planarity seem to be the main reason for the disturbance in self‐assembling. The extra rotation around the azo bonds in the Trypan Blue molecule is slightly extorted by the close proximity of sulfonic groups. This may also be the direct cause of the observed deviation from symmetry in the molecule of this dye. The results confirm that the self‐assembling capability of the compounds studied correlates with the capacity to complex proteins, supporting the idea that supramolecularity may create specific ligation properties.

Force-field parametrization and molecular dynamics simulations of Congo red

Congo red, a diazo dye widely used in medical diagnosis, is known to form supramolecular systems in solution. Such a supramolecular system may interact with various proteins. In order to examine the nature of such complexes empirical force field parameters for the Congo red molecule were developed. The parametrization of bonding terms closely followed the methodology used in the development of the charmm22 force field, except for the calculation of charges. Point charges were calculated from a fit to a quantum mechanically derived electrostatic potential using the CHELP-BOW method. Obtained parameters were tested in a series of molecular dynamics simulations of both a single molecule and a micelle composed of Congo red molecules. It is shown that newly developed parameters define a stable minimum on the hypersurface of the potential energy and crystal and ab initio geometries and rotational barriers are well reproduced. Furthermore, rotations around C-N bonds are similar to torsional vibrations observed in crystals of diphenyl-diazene, which confirms that the flexibility of the molecule is correct. Comparison of results obtained from micelles molecular dynamics simulations with experimental data shows that the thermal dependence of micelle creation is well reproduced.