BACKGROUND: Antibodies are prototypes of multimeric proteins and consist of 
structurally similar domains. The two variable domains of an antibody (VH and 
VL) interact through a large hydrophobic interface and can be expressed as 
covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv 
fragments after long-term denaturation in guanidinium chloride is known to be 
slow. In order to delineate the nature of the rate-limiting step, the folding of 
the scFv fragment of an antibody after short-term denaturation has been 
investigated.
RESULTS: Secondary structure formation, measured by H/D-exchange protection, of 
a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium 
chloride was shown to be multiphasic. NMR analysis shows that an intermediate 
with significant proton protection is observed within the dead time of the 
manual mixing experiments. Subsequently, the folding reaction proceeds via a 
biphasic reaction and mass spectrometry analyses of the exchange experiments 
confirm the existence of two parallel pathways. In the presence of cyclophilin, 
however, the faster 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 intermediate, which shows amide-proton 
exchange protection, is independent of proline isomerization. Subsequently, a 
proline cis-trans isomerization 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 
efficiently catalyzed by cyclophilin.