Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

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.

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. The Fab and Fc fragments in IgG molecules are thermodynamically practically independent subunits in the pH region studied. 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. 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.

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