Paweł Spólnik

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.

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 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.

Formation of amyloid-like aggregates through the attachment of protein molecules to a Congo red scaffolding framework ordered under the influence of an electric field

This study describes a technique which makes it possible to introduce the amyloid-like order to protein aggregates by using the scaffolding framework built from supramolecular, fibrillar Congo red structures arranged in an electric field. The electric field was used not only to obtain a uniform orientation of the charged dye fibrils, but also to make the fibrils long, compact and rigid due to the delocalization of pi electrons, which favors ring stacking and, as a consequence, results in an increased tendency to self-assemble. The protein molecules (immunoglobulin L chain lambda, ferritin) attached to this easily adsorbing dye framework assume its ordered structure. The complex precipitating as plate-like fragments shows birefringence in polarized light. The parallel organization of fibrils can be observed with an electron microscope. The dye framework may be removed via reduction with sodium dithionite, leaving the aggregated protein molecules in the ordered state, as confirmed by X-ray diffraction studies.

The Structure and Function of Living Organisms

The systemic approach adopted in this work focuses on generic biological phenomena. The goal is to explain the strategies employed by biological systems by invoking fundamental concepts in physics, chemistry and information theory. Structure and function is the first of five chapters which make up the book.

The use of the Congo red-related dye DBACR to recognize the heavy chain-derived abnormality of myeloma immunoglobulins

Abstract.IntroductionThe aim of this study was to differentiate heavy and light chain-derived instability of monoclonal myeloma immunoglobulins by complexation of matched supramolecular dyes. These are composed of several micellar pieces of self-assembled dye molecules which may penetrate the protein interior of the binding locus with polypeptide chains. These dyes were used to elicit, by precipitation, the postulated higher aggregation tendency of the heavy chain derived from its higher hydrophobicity.Materials and MethodsAgarose gel electrophoresis was used to create conditions for dye complexation and to reveal the precipitation.ResultsCongo red derivatives with aromatic ring substitutes, BACR and DBACR, of increased penetrating capability were chosen to provoke the precipitation of abnormal immunoglobulins by displacing association-prone polypeptide chains from the protein interior.ConclusionsThe results of this study confirm the heavy chain-related propensity of some monoclonal immunoglobulins to aggregate and precipitate. The simplicity of the technique may improve clinical diagnosis and facilitate predictions of disease complications.

Information—its Role and Meaning in Organisms

Information is necessary in regulatory mechanisms which maintain a steady state of activity in individual cells as well as the whole organism. This state corresponds to a genetically encoded program. Without regulation biological processes would become progressively more and more chaotic. In living cells the primary source of information is genetic material. Studying the role of information in biology involves signaling (i.e. spatial and temporal transfer of information) and storage (preservation of information).

Energy in Biology—Demand and Use

From the point of view of energy management in biological systems, a fundamental requirement is to ensure spontaneity. Process spontaneity is necessary since in a thermodynamically open system—such as the living cell—only spontaneous reactions can be catalyzed by enzymes. Note that enzymes do not, by themselves, contribute additional energy. Spontaneity of biological processes may be expressed by the following correlation: