Human myeloma immunoglobulins of the fourth subclass (IgG4 MAM) contain a fraction with different properties of CH2 domains.

A long-lived metastable minor fraction has been detected and characterized in myeloma protein IgG4 MAM by hydro- and thermodynamic methods. The sedimentation constants of the minor and the major protein fractions are different. The stability of the two CH2 domains in the minor fraction varies. The unique characteristics of these IgG4 MAM conformers arise from the fact that on exchange of the heavy chains between IgG4 molecules, in some of them only one noncanonical bond Cys226-Cys229 is formed in the central part of the "hinge region" instead of two canonical interchain disulfide bonds Cys226-Cys226 and Cys229-Cys229. This leads to asymmetric structure of the IgG4 MAM molecules.

[Correlation between macro- and micro-stability ch2 domains of human IGG2 and their biological activity. 1. Ansalysis the calorimetric and optical melting curves].

Human myeloma immunoglobulin second subclass LOM and SIN, their Fc fragment and firstly obtained hFc fragment in which there is not only low portion of the hinge region, but also its core portion (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys), have been studied by number of physical methods (scanning calorimetry, fluorescence spectroscopy, analytical centrifugation). Joint analysis of calorimetric and optical melting curves revealed that only first (low-temperature) heat absorption peak at all the melting curves corresponds to the melting of the two CH2 domains. It was shown that CH2 domains of intact IgG2 are destabilized relative to those domains in hFc and Fc fragments.

Destabilization of CH2 domains in intact IgG2 is accompanied by reduced ability to inhibit complement system factor C1.

Fc fragments (hFc) of human myeloma IgG2 proteins LOM and SIN having core hinge (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys) were first obtained by a modified proteolytic procedure. The thermostability of CH2 domains inside of standard Fc, hFc fragments, and intact IgG2 LOM and SIN was studied by fluorescence spectroscopy. It was found that CH2 domains of intact IgG2 are destabilized. The destabilization is accompanied by reduced ability of IgG2 to inhibit the activation of complement system by classical pathway. This could be due to the decrease in the affinity of CH2 domains to factor C1q.

Thermodynamic, conformational and functional properties of the human C1q globular heads in the intact C1q molecule in solution.

Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) were studied with the experimental approaches, which allow investigating these properties in the intact hC1q molecule in solution. Surprisingly, the scanning calorimetry data reveal a low level of cooperativity of interactions between the hgC1q A, B and C domains even at a neutral pH area. Ionization of His residues due to acidification of the medium at the pH range from 6 to 5 or the chemical modification of His residues completely abolishes the cooperative interactions between the domains without significant effect on their conformation. The thermodynamic data provide evidence that the hgC1q module is composed of three structurally independent A, B and C globular domains characterized by the practically identical thermal stability and very similar enthalpy of melting. The spectroscopic studies and modification with 2-oxy-5-nitrobenzylbromide (ONBB) indicate that Trp residues in the hgC1q A and C domains are accessible to the solvent that has been confirmed by the hgC1q crystal structure solved and refined to 1.9 A. The modification of Trp residues significantly affects the complement-dependent cytotoxicity without noticeable effect on the hC1q conformation. These data provide evidence that Trp residues are the components of immunoglobulin-binding sites both in the hgC1q A and C domains.

Effect of hinge region state on interaction of human IgG3 with the complement system.

Covalently cross-linked rod-like dimers of human myeloma IgG3 are more efficient in the inhibition of the complement system reaction than compact dimers. This correlates with an increase in the stability of CH2 domains of the immunoglobulin in the state with the rod-like hinge region.

Three states of the pFh-fragment (Hinge region) from human myeloma IgG3 Kuc with native-like secondary structure.

The structure of the pFh-fragment (hinge region) from human myeloma IgG3 Kuc (the third subclass immunoglobulin) was studied by hydrodynamic methods in the pH range from 3.0 to 8.0. The pFh-fragment was found to occur in three states, each with a high content of the secondary structure: a rod-like state at pH < or = 4.0, a "molten globule" state at pH 4.2-5.5, and the native state at pH 7. 5-8.0.

Investigation of the cooperative structure of Fc fragments from myeloma immunoglobulin G.

The cooperative structure of Fc fragments prepared from myeloma human IgG1 was studied using scanning microcalorimetry and fluorescence at pH 4.2-8.0. It was shown that the first to be melted are CH2 domains whose interaction with each other is rather weak, while that with CH3 domains is strong. Then CH3 domains which form a single cooperative block are melted. The data for the structure of the Fc fragment in solution agree with the X-ray data according to which the interaction between CH2 domains is mediated by the carbohydrate moiety while the two CH3 domains are strongly associated. The presence of intensive CH2-CH3 interaction is a distinctive feature of the state of the Fc fragment in the given pH region as compared to that at pH <4.1 [Tischenko, V. M., et al. (1982) Eur. J. Biochem. 126, 517-521; Ryazantsev, S., et al. (1990) Eur. J. Biochem. 190, 393-399]. First, cis interactions greatly increase the free energy of the native structure stabilization in CH2 domains. Second, they decrease this energy for CH3 domains when compared to the state of the latter at pH 3.8 or within the Fc' fragment (the dimer of CH3 domains). The temperature and enthalpy of melting of CH2 domains coincide in all the samples studied despite heterogeneity of the carbohydrate moiety. Thus, it may be postulated that the conservative part of CH2 domains makes a cardinal contribution to the interaction of these domains with the carbohydrate moiety.

Thermodynamic studies of the collagen-like region of human subcomponent C1q. A water-containing structural model.

Thermal transitions of Clq were investigated by methods of differential scanning calorimetry, circular dichroism and fluorescence. The melting curves of Clq display two pronounced heat absorption peaks with enables determination of the thermodynamic parameters characterizing each transition. The low temperature peak was assigned to melting of the Clq collagenous part. Analysis of the data has revealed unusual, as compared with the monomeric collagen molecules, thermodynamic features of the Clq collagenous part: (1) higher thermal stability strongly dependent on pH; (2) less linear co-operative regions; and (3) a noticeable change in the partial specific heat capacity (delta Cp) in contrast to both the monomeric collagen and the collagen fibrils. This unusually large delta Cp value suggested a conclusion that the fibril-like endpiece of Clq may have a cavity filled with ice-like ordered water molecules.

Correlation of the character of intramolecular melting with digestibility by pepsin in precipitating and non-precipitating pig anti-Dnp antibodies.

Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were investigated by differential adiabatic scanning microcalorimetry in the pH range 3.7-4.5. The partial heat capacity functions obtained revealed a notable difference between the two antibody types when the analysis was done at pH 4.5 or 4.0. The transition observed at temps around 50 degrees C in a non-precipitating antibody was absent in a precipitating antibody. Under analogous conditions, pH 4.5, the precipitating antibody was fully resistant to pepsin, while the non-precipitating antibody yielded appropriate F(ab')2 and pFc'fragments. At pH 3.7 no substantial difference in the partial heat capacity function could be observed between the two antibody types. Below pH 4.0 the precipitating antibody became susceptible to peptic cleavage and yielded fragments of the same general character as the non-precipitating antibody. This finding lends support to the view that the structural block in immunoglobulin G that melts first, i.e. at temps near 50 degrees C, is the CH2 domain or a part of it.

A thermodynamic study of cooperative structures in rabbit immunoglobulin G.

The intramolecular melting of rabbit immunoglobulin G and its proteolytic fragments has been studied by scanning microcalorimetry in the acidic pH region. By thermodynamic analysis of the heat capacity function the following conclusions have been made. 1. The Fab and Fc fragments in IgG molecules are thermodynamically practically independent subunits in the pH region studied. 2. The Fab fragment exhibits three cooperative transitions at melting. Two of these transitions are explained assuming that equivalent domains in this fragment are paired, forming two cooperative blocks, while the third transition seems to correspond to the disruption of contacts between these cooperative blocks. 3. Fc and pFc' fragments exhibit four and two cooperative transitions, respectively. These transitions can be explained only by assuming that each of the structural domains of these fragments consists of two subparts and that the pairs of equivalent subparts are merged into the cooperative blocks thus forming four cooperative blocks in the Fc fragment.