PARALLEL PATHWAYS IN THE FOLDING OF A SHORT-TERM DENATURED SCFV FRAGMENT OF AN ANTIBODY

Background: Antibodies are prototypes of multimeric proteins and consi st of structurally similar domains. The two variable domains of an ant ibody (V-H and V-L) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragment s. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate th e nature of the rate-limiting step, the folding of the scFv fragment o f an antibody after short-term denaturation has been investigated. Res ults: Secondary structure formation, measured by H/D-exchange protecti on, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis sh ows that an intermediate with significant proton protection is observe d within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectr ometry analyses of the exchange experiments confirm the existence of t wo parallel pathways. in the presence of cyclophilin, however, the fas ter of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin. Conclusions: The formation of an early interme diate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerizatio n reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficie ntly catalyzed by cyclophilin.