ASSOCIATION OF ANTIBODY CHAINS AT DIFFERENT STAGES OF FOLDING - PROLYL ISOMERIZATION OCCURS AFTER FORMATION OF QUATERNARY STRUCTURE

The folding pathways of multi-domain proteins are still poorly underst ood due to the complexity of the reaction involving domain folding, as sociation and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the het ero-dimeric protein from the completely denatured, oxidized state comp rises the pairwise association of the two domains of each chain with t hose of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linke d via a cystine bond (Fab) and that of a mutant lacking this covalent Linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragmen t is a slow process, which can be described by a single exponential te rm. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/ -cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This im plies that, during Fab reactivation, Fd associates with the light chai n at the stage of an earlier folding intermediate, thus eliminating th e additional slow folding step of the light chain observed with Fab/-c ys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the conf iguration of at least one prolyl-peptide bond. Using the different sub strate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsib le for the observed slow folding phase after association, although thr ee other proline residues adopt a cis configuration in the native prot ein. Furthermore, the data suggest that in the case of the Fab fragmen t, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary stru cture determines the cis-configuration of the prolyl residue in Fd inv olved in the rate-limiting folding reaction.