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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 beta-structure and disulfide bonds as well as the participation of natural ligand in folding process seem to be necessary.

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 beta-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 lambda 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 lambda chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.

The model adopting the three-dimensional Gauss function to express the hydrophobicity distribution in proteins is presented in this paper. The tendency to create the hydrophobic center during protein folding is expressed in form of an external force field of the form of three-dimensional Gauss function which directs the folding polypeptide to locate the hydrophobic residues in a central part of the molecule and hydrophilic ones exposed toward the molecular surface. The decrease of the differences between hydrophobicity distribution as it appears at each step of the folding simulation and the expected hydrophobicity distribution (three-dimensional Gauss function) is the convergence criterion together with traditional non-bonding interaction optimization. The model was applied to fold the hypothetical membrane protein (target protein in CASP6) TA0354_69_121 from Thermoplasma acidophilum.

Methods for biological function recognition in silico appeared to be useful also for identifying characteristics of structure-to-function relations. The introduction of a three-dimensional Gauss function was assumed to represent the hydrophobic core in a protein molecule. The discrepancy between idealized "fuzzy oil-drop" and the observed one in real proteins appeared to be localized in the ligation site or in the area of biological function related part of protein molecule. The examples of proteins presented in this paper reveal that the structure-function relation can be evaluated and characterized also using the profile of the difference in value between idealized and real hydrophobicity distribution along the polypeptide chain. The specificity of particular polypeptide chain fragments in respect to their biological function and their specific participation in active site creation is discussed in this paper. The scale allowing comparison of different proteins in respect to their ligand-binding sites characteristics is introduced.

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 beta-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 beta-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., (Copyright 2005 Wiley-Liss, Inc.)

Congo red, a dye of high self-assembling tendency, has been found to form complexes with proteins by adhesion of the ribbon-like supramolecular ligand to polypeptide chains of beta-conformation. Complexation is allowed by local or global protein instability, facilitating penetration of the dye to the locus of its binding. At elevated temperatures, L chain lambda of myeloma origin was found to form two distinct complexes with Congo red, easily differentiated in electrophoresis as slow- and fast-migrating fractions, bearing four- and eight-dye-molecule ligands, respectively, in the V domain of each individual chain. The slow-migrating complex is formed after displacement of the N-terminal polypeptide chain fragment (about 20 residues) from its packing locus, thereby exposing the entrance to the binding cavity. In this work the formation and stability of this complex was studied by molecular dynamics (MD) simulations. The effect of three- and five-molecule ligands introduced to the site binding the dye was also analyzed in an attempt to understand the formation of fast-migrating complexes. The wedging of the ligand containing five dye molecules, hence longer than established experimentally as the maximum for the slow-migrating complex, was found to generate significant structural changes. These changes were assumed to represent the crossing of the threshold on the way to forming a fast-migrating complex more capacious for dyes. They led to almost general destabilization of the V domain, making it susceptible to extra dye complexation. Theoretical studies were designed in close reference to experimental findings concerning the number of dye molecules in the ligand inserted to the site binding the dye, the location of the site in the domain, and the conditions of formation of the complexes. The results of the two kinds of studies appeared coherent.

Estimation of structure predictability for a particular protein is difficult. Many methods estimate it in an a posteriori system evaluating the final, native protein structure. The SPI scale is intended to estimate the structure predictability of a particular amino acid sequence in an a priori system. A sequence-to-structure library was created based on the complete Protein Data Bank. The tetrapeptide was selected as a unit representing a well-defined structural motif. The early-stage folding structure (a model of which was presented elsewhere) was taken as the object for protein structure classification. Seven structural forms were distinguished for structure classification. The degree of determinability was estimated for the sequence-to-structure and structure-to-sequence relations particularly interesting for threading methods. A comparative analysis of the SPI and Q7 scales with the commonly used SOV and Q3 scales is presented. The complete contingency table, supplementary materials and all the programs used are available on request.

The conformational sub-space oriented on early-stage protein folding is applied to lysozyme folding. The part of the Ramachandran map distinguished on the basis of a geometrical model of the polypeptide chain limited to the mutual orientation of the peptide bond planes is shown to deliver the initial structure of the polypeptide for the energy minimization procedure in the ab initio model of protein folding prediction. Two forms of energy minimization and molecular dynamics simulation procedures were applied to the assumed early-stage protein folding of lysozyme. One of them included the disulphide bond system and the other excluded it. The post-energy-minimization and post-dynamics structures were compared using RMS-D and non-bonding contact maps to estimate the degree of approach to the native, target structure of the protein molecule obtained using the limited conformational sub-space for the early stage of folding.

The postulated intramolecular signaling in immunoglobulins generated by antigen binding has been controversial for years. The high heterogeneity of immune complexes as signaling systems and the requirement of the immobilized antigen form for efficient triggering of effector activity is likely the reason for the lack of clarity. Here we present new evidence supporting the notion of intramolecular signaling, based on the use of supramolecular dyes that bind to signal-derived specific sites in immunoglobulins.

Monoclonal myeloma proteins often have an abnormal, unstable structure, and tend to aggregate with fatal clinical consequences. A method for early clinical identification of this aggregation tendency is impatiently awaited. This work proposes the use of supramolecular dyes as specific ligands to reveal protein instability. Disclosure of excessive polypeptide chain flexibility in unstable monoclonal proteins, leading to increased susceptibility to penetration by foreign compounds, appeared possible when new supramolecular Congo red-derived dyes with different protein-binding capabilities were used for complexation. Two basic protein instability levels, local and global, were differentiated by comparing the extent of protein loading with dye and the subsequent electrophoretic migration rate of the complexes. A simple electrophoretic test is proposed for assessment of the instability of monoclonal proteins in clinical conditions.

A probability calculus was used to simulate the early stages of protein folding in ab initio structure prediction. The probabilities of particular phi and psi 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 phi and psi angles to be predicted. It was found that the amount of information needed to predict phi and psi angles with 5 degrees 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., (Copyright 2004 Wiley-Liss, Inc.)

Supramolecular micellar structures have been proposed as carriers in aim-oriented drug transportation to a target marked by specific immune complexes. In this study, the self-assembling dye Congo red was used as a model supramolecular carrier and its accumulation in the target was studied in vivo. The target was created in vivo as the local specific inflammation provoked by subcutaneous injection of antigen to the ear of a previously immunized rabbit. The color caused by accumulation of Congo red after its intravenous injection was registered by pictures of the ear with suitably filtered visible light shining through it to distinguish Congo red against the background color of hemoglobin. The results confirmed the expected accumulation and retention of Congo red in the inflammation area marked by deposits of specific immune complexes. The role of albumin and its possible interference with transportation of drugs through the blood by supramolecular carriers was also subjected to preliminary examination. The results revealed that albumin collaborates rather than interferes with drug transportation; this is another factor making the use of supramolecular carriers for aim-oriented chemotherapy highly promising.

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.

The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible., (Copyright 2003 Wiley Periodicals, Inc.)

Background: Frequently observed structural deviations of myeloma-derived immunoglobulins affect polypeptide chain packing and domain stability, enhancing their tendency to aggregate, with all the clinical consequences. Congo red complexation with myeloma immunoglobulins is proposed in this work as a general test to disclose the instability of these proteins. The large ribbon-like supramolecular ligands of Congo red form complexes with proteins by adhesion to beta-conformation polypeptide chains, if allowed to make contact with their backbone interfaces. This can occur in the case of myeloma-derived immunoglobulins with deficient polypeptide chain packing., Material/methods: Specially adapted two-dimensional agarose electrophoresis of serum proteins, which allows the transient contact of Congo red and serum proteins during migration, was used to reveal the presence of protein components amenable to ligand penetration and binding. The combination of electrophoresis and Congo red binding to proteins permits the removal of loosely attached dye and evaluation of the effective complexation properties of the immunoglobulin fraction directly in the serum., Results: Comparative studies of dye complexation with two L chains having different reactivities with Congo red confirmed that dye binding depended on protein instability in the conditions used. Myeloma proteins revealed different binding capabilities in the test used here., Conclusions: The complexes formed by the supramolecular dye Congo red with myeloma immunoglobulins differ in stability. Those of high stability indicate the abnormal protein structure thought to produce clinical symptoms. This work proposes an easy technique to differentiate the stability of complexes.

Motivation: The expanding protein sequence and structure databases await methods allowing rapid similarity search. Geometric parameters-dihedral angle between two sequential peptide bond planes (V) and radius of curvature (R) as they appear in pentapeptide fragments in polypeptide chains-are proposed for use in evaluating structural similarity in proteins (VeaR). The parabolic (empirical) function expressing the radius of curvature's dependence on the V-angle in model polypeptides is altered in real proteins in a form characteristic for a particular protein. This can be used as a criterion for judging similarity., Results: A structural comparison of proteins representing a wide spectrum of structures was assessed versus sequence similarity analysis based on the genetic semihomology algorithm. The term 'consensus structure', analogous to 'consensus sequence', was introduced for the serpine family., Availability: Semihom-sequence comparison freely available on request from J. Leluk. VeaR-structural comparison freely available on request from I. Roterman.

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 supramolecular dye Congo red was used to check whether monocyte activation may be mediated by a torsion-dependent mechanism preventing transduction of weak random signals in cell contacts in a way corresponding to the discrimination mechanism found in complement fixation by immune complexes. Tight cell-cell contacts generating torsional effects may be expected to produce alteration of receptor structure, making them accessible for binding of supramolecular dyes. In this study, Congo red was used to observe the binding accessibility of (1) monocytes (human) induced by contact with cancer cells (HCV29T, human), (2) monocytes (mouse) stimulated by interaction with heat-aggregated IgG and (3) monocytes (mouse) activated by rosetting in the presence of an SRBC-anti-SRBC system. Microscopic studies confirmed the activation of monocytes manifested by their clustering and Congo red binding, but only tightly clustered cells appeared to attach the dye on the surface. Usually not the whole cell surface is found to be engaged in dye complexation. Staining occurs predominantly on the interfaces of reacting cells, making probable the suggestion that cell adhesion receptors are involved in dye binding. The cells in the central areas of tight clusters undergo accelerated death. In the presence of Congo red they are easily recognized as intensely fluorescent. The characteristic localization of dead cells in the central area of clusters indicates that death is not random but results from cell activation. The role of Congo red in this process remains to be clarified. The staining characteristics of monocytes after application of Congo red probably discloses the initial step in signal transduction generated by torsional movements in receptor proteins.

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.

Background: The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions., Material and Methods: The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand., Results: Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly., Conclusions: The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon-like self-assembled form of the dye.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.

Two experiments, an off-line acceptability judgment study and an on-line self-paced reading experiment, were conducted to tackle the question of locality-based preferences in sentence processing. The material consisted of German verb-final sentences containing a relative clause that was either host adjacent or extraposed. While the off-line data seem to reflect locality based integration cost, the on-line data revealed a highly different picture. It is argued that, in the present constructions at least, locality is relevant for production rather than perception. In perception, heads can be anticipated through incremental integration of their arguments.

Two accounts of relative clause attachment will be discussed, the case-matching hypothesis proposed by Sauerland and Gibson (1998) and the attachment-binding dualism (Hemforth et al., in press a, b). While the case-matching hypothesis predicts that relative clauses are preferentially attached to NPs whose case matches that of the relative pronoun, attachment binding predicts that NPs are preferentially attached to the most salient host, that is NP1 in constructions with two NPs. We conducted two off-line studies, one sentence completion task and one magnitude estimation experiment using subject (nominative pronoun) and object (accusative pronoun) relative clauses that can be attached to either of the two nouns in a complex subject (NP1 = nominative, NP2 = genitive) or object NP (NP1 = accusative, NP2 = genitive). While attachment binding predicts an across-the-board NP1 preference, the case-matching hypothesis predicts an NP1 prefence only in the case of subject (object) NPs followed by subject (object) relative clauses. The results of both experiments provide evidence for attachment binding and against case matching.

The lyotropic liquid crystal dye-Congo Red was used as a carrier in a model immunotargeting system constructed from sheep red blood cells (SRBC) representing the antigen target and rabbit IgG anti-SRBC as the specific driving immunoglobulin. Rhodamine B and Hemin stains were chosen as example chemicals carried to the target. The carried stains were introduced to the micellar organization of Congo Red by intercalation. Preserving its supramolecular organization, Congo Red binds spontaneously and selectively to antibodies that have altered structure extorted by interaction with the antigen in the immune complex. The functionality of the studied immunotargeting model was verified by fluorescence and electron microscopy. The results indicate that the supramolecular nature of protein ligands offers new ligation capabilities possibly useful for carrying stains or drugs in immune-oriented systems.

The study concerns the problem of selective binding of Congo Red dye to serum proteins. The dye-binding structural motifs were assumed to appear in proteins as the result of local disruptions of tertiary interaction associated with biological activities. Functional structures of serum proteinase inhibitors and haptoglobins induced by interaction of these proteins with their natural substrates were found to bind Congo Red. Soluble immune complexes formed by bivalent hapten-bridged anti-TNP rabbit antibodies also appeared able to interact with the dye. The possible use of these effects for diagnostic analysis was suggested.

Lyotropic liquid crystal mesophases, represented in this work by Congo Red bis azo dye solution, were proposed as systems to carry drugs to immuno-selected targets. The possible use of Congo Red for this aim arises from its liquid crystalline features, enabling it to attach to immune complexes as polymolecular, ordered conglomerates and simultaneously to incorporate many organic compounds into its mesophase, largely independent of the water with a specific but adaptive molecular organization. Molecules with planar rigid structure, and/or large hydrophobic fragments, especially those with a positively charged group in the molecule, are found to be incorporated best. Rhodamine B, Rhodamine 6G and adriamycine, which have the assumed binding features, were tested as model compounds and were found to be readily engaged into Congo Red mesophase. The effect of hydrophobicity on ligand binding was evaluated following the incorporation of homologic 10, 12, 14, 16 carbon chain organic acids and the effect of charge using small mobile tandem molecules of pI differing by a few pH units (lysine-norleucine, tyrosine-tyramine). Positive charge seems to affect binding especially by influencing the organization of molecules and the shape of the micelle simultaneously. Congo Red immobilized to heat-aggregated immunoglobulins and antibodies in the immune complex was found to retain its binding ability, confirming its possible usefulness for drug transport.

Molecular dynamics simulation was used to identify the conformational alterations in the Fab fragment (Kol), driven by heating at 300, 320, and 340 K. Comparison of heat-modified VH,CH1 and VL,CL domain structures with the corresponding crystal conformations revealed specific differences, most definitely expressed in the CH1 domain. These are dislocations of predominantly peripheral peptide loops exposed to the V-C interdomain interface, comprising in particular the 175-185 amino acids of the heavy chain, as well as the 112-123 amino acids of the interdomain hinge fragment. The deviations, limited to peripheral domain regions at 300 and 320 K, spread at 340 K. The resulting relaxation of the tertiary packing in the protein (including the hydrophobic core) initiates global melting of the domain. These theoretical results were supported by experimental findings concerning penetration and binding of dyes (Congo Red, Trypan Blue, ANS) to the protein. Packing relaxation of the CH1 domain is turned on after disruption of the specific peptide arrangement formed at the V-C interdomain interface basically at the hinge portion (117-122) and at fragments of adjacent peptide loops (149-154, 171-179) of CH1 origin, most probably playing the role of a switching mechanism. The dislocations also comprise the 131-141 amino acids of the loops accommodated at the CH1-CH2 interface. However, the lack of crystallographic data concerning the Fab-Fc interface limits discussion of this effect to speculation. It was concluded that the unconcerted movements of the V and C parts of the Fab fragment are an intrinsic driving mechanism, introducing structural alterations into the C domains. It is suggested that the domain relaxation, induced by heating or mechanical constraints, allows for intermolecular interactions, affecting in this way the stability of the immune complex.

Azo dyes (Congo Red, Evans Blue, Trypan Blue) were used to study the relation between antigen binding and effector activity in SRBC rabbit anti-SRBC IgG system. Antigen binding was tested in this study by agglutination and effector activity by complement fixation. Antibodies induced by the antigen binding appeared to become structurally susceptible for interaction with the dye. The dyes cause the enhancement of antigen binding by antibodies and inhibition of their effector activity. The intensity of both effects caused by binding the dye seems correlated. The increase of antibody concentration makes the effector activity of immune complexes more resistant to dye inhibition indicating that the influence of antigen binding may be exerted on assembling of antibody in the immune complex rather than on structural modulation of Fc. Also the analysis of dye effects on antibody derivatives deprived of effector activity (Fab)2, IgG with split interheavy chain disulphides supplied evidence that the induced by the antigen long distance structural effects may play the role of immune signal by influencing the assembling of antibodies and stability of immune complex.

The mechanism of binding of azo dyes (bis azo) to immunoglobulin G of altered conformation, induced by heating or interaction with antigen was analysed in this work. Azo dyes: Congo Red, Evans Blue and Trypan Blue were selected for these studies. The molecules of Congo Red and Evans Blue associate readily in water and exist as polymolecular micellar species of liquid crystalline organization. Such organization of molecules appeared necessary for these dyes to interact with antibodies and to affect the formation of immune complex. It was proved by studying the properties of isomeric dyes Evans Blue and Trypan Blue, whose ability to form polymolecular conglomerates in water differs, being high for Evans Blue and low for Trypan Blue. The dyes seem to influence the formation of the immune complex generally by interacting with individual immunoglobulin molecules. The rate of exchange of dye molecules in conglomerate bound to protein varies and is the lowest for the portion of molecules which are engaged directly in the complex with protein. Electron microscopic studies also confirmed the polymolecular form of the dye in the dye-protein complex. The computational simulation of dye-dye and dye-protein interaction was performed in a model system. The micelle in the system was represented by three molecules of Congo Red. The peptide loop composed of amino acids 68-88 originating from VL IgG domain, was selected to represent protein. Amino acid side chains of this fragment were reduced to C beta. The best fitness was found for peptide chains of twisted beta conformation and independently optimized conformation of the dye in a form of "twisted ribbon" micelle. It was concluded that the IgG domains become accessible for penetration of the dye after being relaxed in the result of heating or interaction with the antigen.

The azo dyes were found to react with antibodies aggregated in immune complex in a similar way to heat-aggregated IgG. The whole micelles of the dye, instead of single molecules, are fixed to antibodies. In a consequence, the number of dye molecules, determined per one antibody molecule, differs but may be as large as 50-60. The dye, bound to antibody complexed with antigen, enhances its affinity to antigen. The increase of affinity, expressed as the relation of changed by the dye association constants to its initial value, was found to be 6.27 x 10(8):1. The corresponding energy change, calculated from this value, equals 11.97 kcal/mol. The resulting amount of antibodies, bound to antigen, is essentially enlarged. The enhancement effect does not seem to depend on the number of antibodies bound to the red cell up to about 3 x 10(4) molecules. It indicates that in this range a single antibody binding may be affected by the dye. The situation changes at growing antibody densities in the complex when the dye-antibody network, formed by crossbridged molecules, additionally increases the stability of the immunoglobulin molecules engaged in the complex.

Sodium thiosulfate was used to enhance in vivo the polymerization of myeloma IgM, deficient in disulfide cross-links. The therapy sharply decreased the amount of low molecular weight IgM fractions, while increasing the serum content of molecules of higher molecular weight. The degree of disulfide cross-linking in IgM increased under the influence of thiosulfate. The rate of secretion into the serum and urine of some membrane-related glycopeptides and species rich in sialic acid was reduced. Also, the discharge of L chains to the urine was lowered during the thiosulfate trial. All these changes were attributed to enhancement of disulfide-interchanging enzyme activity by thiosulfate.

The mechanism of IgG heat aggregation was studied using IgG aggregates complexed with azo dyes to increase their solubility and stability. Heat dependent and heat independent steps of aggregation were differentiated. On heating IgG at the dye concentration exceeding 100 times that of protein, mainly dimers are formed, as judged from ultracentrifugation and chromatographic analysis, whereas high molecular weight derivatives appear at room temperature when the protein/dye ratio is decreased. The analysis of spectral changes following either the attachment or removal of the dye from IgG aggregates implies that only a part of the dye molecules is bound firmly and directly to the protein binding sites. These dye molecules which are easily removed by adsorption to cellulose or reduced by dithionate but migrate together with IgG aggregates on chromatography and electrophoresis, are supposed to constitute that part of the micelle which extrudes from the binding site and, hence, is fixed indirectly to protein. Various proteins with predominant beta-structure were also found to bind azo dyes when heated.

It has been suggested that the low disulfide interchange activity in cells producing IgM causes the excretion of immunoglobulin molecules with deficient disulfide cross-linking. IgM material found in the serum formed aggregates which were stabilized largely by noncovalent bonds. Sedimentation coefficients of these aggregates ranged from 11 to 19 S and were similar to sedimentation coefficients calculated for molecules composed of 2, 3, 4 and 5 IgM subunits. A fraction corresponding to a single IgM subunit with a sedimentation coefficient 7.1 S was also present in the serum. The molecular weight of this fraction, determined in mild conditions by Sephadex gel filtration, appeared higher than that predicted for IgMs, reaching the value of 270 000-280 000. The evident increment of molecular weight, compared to a single IgM subunit, was attributed to glycopeptides of sedimentation coefficient 2 S, which form the complex with IgMs molecules. These glycopeptides were also present in aggregates consisting of more than one IgM subunit. The number of IgM subunits participating in aggregates and their molecular weights appeared to depend on the ratio of 2 S glycopeptides to IgM material. With the increase of this ratio the molecular weight of the aggregates lowered and vice versa. Electrical charge and some physico-chemical properties of the aggregates were also significantly affected by the presence of glycopeptides, 2 S glycopeptides were supposed to be the basement membrane related material. Suggestions concerning the interpretation of the phenomenon are presented and discussed.

Excessive production of IgM in macroglobulinemia is accompanied frequently by secretion of proteins and peptides which are normally absent from biological fluids. These proteins and peptides are probably secreted by cells producing IgM. Three types of peptides were isolated and their basic properties are described. One peptide showed calcium-binding properties, and the other two peptides were bound to secreted IgM immunoglobulin.

An electrochromatographic method for the analysis of sera is described with particular design to studies of abnormal proteins. The method allows a simultaneous but independent evaluation of molecular weight and charge related migration of proteins. A modification which may improve the analysis of immunoglobulins by increasing their electrophoretic mobility is described.

The arrangement of peptides which form the hinge region in Fab-Fc recombinant immunoglobulins, restored artificially from Fab and Fc fragments was approached by computation. The architecture of this region in the symmetric (Fab')2-Fc derivatives and in the asymmetric (Fab')1-Fc differs to a considerable extent. In (Fab')2-Fc species but not in (Fab')1-Fc the preferable arrangement appeared to be stabilized predominantly by the mutual interaction of symmetric hinge peptides. It was concluded that the resulted by this interaction rotational restrictions may eventually induce the structural transformations in the molecule, influencing the effector activity of Fc.

The structural alteration associated with the aggregation of heated immunoglobulin molecules and the triggering of their effector reactivity was studied using soluble aggregate derivatives. Large but still soluble aggregate species were obtained by the formation of complexes of heated immunoglobulin molecules with azo dyes - Congo Red or Trypan Blue. Exposed peptide fragments in the area rearranged on heating were identified by short-lasting proteolysis. The altered area was in this way localized in the Fab portion of the heavy chain. The heavy chain fragment bordered by disulphides which stabilize the domains V and CH1 was indicated as a most probable peptide portion forming the link between the aggregating molecules. The obtained dye-aggregate species retain their effector activity. The effect of structural alterations in the Fab fragment on the Fc remains unclear.

It was found that intravenous application of thiosulfate increased non-specifically immunoglobulin concentration in mice sera. Thiosulfate stimulated the immune response to T-dependent antigens.