Janina Rybarska

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 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.

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.

The indirect generation of long-distance structural changes in antibodies upon their binding to antigen

An allosteric mechanism for the generation of long‐distance structural alterations in Fab fragments of antibodies in immune complexes has been postulated and tested in theoretical and experimental analysis. The flexing and/or torsion‐derived forces exerted on the elbow region in Fab arms of bivalent antibodies upon binding to antigen were assumed to drive the disruption of hydrogen bonds which stabilize N‐ and C‐terminal chain fragments in V‐domains. This allows an extra movement in the elbow followed by a relaxation in the Fab arm and may generate long‐distance effects if, in particular, the structural changes are generated asymmetrically involving one chain of the Fab arm only. This mechanism was studied by simulation of molecular dynamics. The local instability in the area involving the site of packing of the N‐terminal chain fragment allows penetration and binding of the supramolecular dye Congo red that hence becomes an indicator of the initiated relaxation process and is also the prospective ligand in studies of designing drugs. The susceptibility to dye binding was observed in complexation of bivalent antibodies only, supplying the evidence that constraints associating the interaction with randomly distributed antigenic determinants drive the local structural changes in the V‐domain followed by long‐distance effects.

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction.

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N‐terminal polypeptide chain, as observed experimentally in a model system—immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N‐terminal fragment and self‐assembled ligand in coupling between V and C domains. Root‐mean‐square distance (RMSD) time profiles show that removal of N‐terminal fragment leads to destabilization of V domain. A micelle composed of four self‐assembled dye molecules stabilizes and fixes the domain. Analysis of root‐mean‐square fluctuation (RMSF) values and dynamic cross‐correlation matrices (DCCM) reveals that removal of N‐terminal fragment results in complete decoupling between V and C domains. Binding of self‐assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small β‐sheet composed of N‐ and C‐terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self‐assembled ligand, bound in the place originally occupied by N‐terminal fragment, is not able to take over the function of the β‐sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain–Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction. Proteins 2005.

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.

The increased flexibility of CDR loops generated in antibodies by Congo red complexation favors antigen binding.

Abstract The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the β-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L λ chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye- enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L λ chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.

Influence of the electric field on supramolecular structure and properties of amyloid-specific reagent Congo red

Among specific amyloid ligands, Congo red and its analogues are often considered potential therapeutic compounds. However, the results of the studies so far have not been univocal because the properties of this dye, derived mostly from its supramolecular nature, are still poorly understood. The supramolecular structure of Congo red, formed by π–π stacking of dye molecules, is susceptible to the influence of the electric field, which may significantly facilitate electron delocalization. Consequently, the electric field may generate altered physico-chemical properties of the dye. Enhanced electron delocalization, induced by the electric field, alters the total charge of Congo red, making the dye more acidic (negatively charged). This is a consequence of withdrawing electrons from polar substituents of aromatic rings—sulfonic and amino groups—thus increasing their tendency to dissociate protons. The electric field-induced charge alteration observed in electrophoresis depends on dye concentration. This concentration-dependent charge alteration effect disappears when the supramolecular structure disintegrates in DMSO. Dipoles formed from supramolecular fibrillar species in the electric field become ordered in the solution, introducing the modified arrangement to liquid crystalline phase. Experimental results and theoretical studies provide evidence confirming predictions that the supramolecular character of Congo red is the main reason for its specific properties and reactivity.