A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

We have studied the equilibrium unfolding and the kinetics of folding and unfolding of an antibody scFv fragment devoid of cis-prolines. An anti-GCN4 scFv fragment carrying a V-L lambda domain, obtained by ribosome display. served as the model system together with an engineered destabilized mutant in V-H carrying the R66K exchange. Kinetic and equilibrium unfolding experiments indicate that the V-H mutation also affects V-L unfolding, possibly by partially destabilizing the interface provided by V-H, even though the mutation is distant from the interface. Upon folding of the scFv fragment, a kinetic trap is populated whose escape rate is much faster with the more stable V-H, domain. The formation of the trap can be avoided if refolding is carried out stepwise. with V-H folding first. These results show that antibody scFv fragments do not fold by the much faster independent domain folding, but instead form a kinetically trapped off-pathway intermediate, which slows down folding under native conditions. This intermediate is characterized by premature interaction of the unfolded domains, and particularly involving unfolded V-H, independent of proline cis-trans isomerization in V-L. This work also implies that V-H should be a prime target in engineering well behaving antibody fragments. (C) 2002 Elsevier Science B.V. All rights reserved.