Irena Roterman

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. Two 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. Molecular 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.

Prediction of functional sites based on the fuzzy oil drop model

A description of many biological processes requires knowledge of the 3-D structure of proteins and, in particular, the defined active site responsible for biological function. Many proteins, the genes of which have been identified as the result of human genome sequencing, and which were synthesized experimentally, await identification of their biological activity. Currently used methods do not always yield satisfactory results, and new algorithms need to be developed to recognize the localization of active sites in proteins. This paper describes a computational model that can be used to identify potential areas that are able to interact with other molecules (ligands, substrates, inhibitors, etc.). The model for active site recognition is based on the analysis of hydrophobicity distribution in protein molecules. It is shown, based on the analyses of proteins with known biological activity and of proteins of unknown function, that the region of significantly irregular hydrophobicity distribution in proteins appears to be function related.

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.

Conformational subspace in simulation of early‐stage protein folding

A probability calculus was used to simulate the early stages of protein folding in ab initio structure prediction. The probabilities of particular ϕ and ψ angles for each of 20 amino acids as they occur in crystal forms of proteins were used to calculate the amount of information necessary for the occurrence of given ϕ and ψ angles to be predicted. It was found that the amount of information needed to predict ϕ and ψ angles with 5° precision is much higher than the amount of information actually carried by individual amino acids in the polypeptide chain. To handle this problem, a limited conformational space for the preliminary search for optimal polypeptide structure is proposed based on a simplified geometrical model of the polypeptide chain and on the probability calculus. These two models, geometric and probabilistic, based on different sources, yield a common conclusion concerning how a limited conformational space can represent an early stage of polypeptide chain‐folding simulation. The ribonuclease molecule was used to test the limited conformational space as a tool for modeling early‐stage folding. Proteins 2004.

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.

Fuzzy-Oil-Drop Hydrophobic Force Field—A Model to Represent Late-stage Folding (In Silico) of Lysozyme

Abstract A model of hydrophobic collapse (in silico), which is generally considered to be the driving force for protein folding, is presented in this work. The model introduces the external field in the form of a fuzzy-oil-drop assumed to represent the environment. The drop is expressed in the form of a three-dimensional Gauss function. The usual probability value is assumed to represent the hydrophobicity distribution in the three-dimensional space of the virtual environment. The differences between this idealized hydrophobicity distribution and the one represented by the folded polypeptide chain is the parameter to be minimized in the structure optimization procedure. The size of fuzzy-oil-drop is critical for the folding process. A strong correlation between protein length and the dimension of the native and early-stage molecular form was found on the basis of single-domain proteins analysis. A previously presented early-stage folding (in silico) model was used to create the starting structure for the procedure of late-stage folding of lysozyme. The results of simulation were found to be promising, although additional improvements for the formation of β-structure and disulfide bonds as well as the participation of natural ligand in folding process seem to be necessary.

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.

Fuzzy oil drop model to interpret the structure of antifreeze proteins and their mutants

Mutations in proteins introduce structural changes and influence biological activity: the specific effects depend on the location of the mutation. The simple method proposed in the present paper is based on a two-step model of in silico protein folding. The structure of the first intermediate is assumed to be determined solely by backbone conformation. The structure of the second one is assumed to be determined by the presence of a hydrophobic center. The comparable structural analysis of the set of mutants is performed to identify the mutant-induced structural changes. The changes of the hydrophobic core organization measured by the divergence entropy allows quantitative comparison estimating the relative structural changes upon mutation. The set of antifreeze proteins, which appeared to represent the hydrophobic core structure accordant with “fuzzy oil drop” model was selected for analysis.