Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment

refolding kinetics of a single-chain Fv (scFv) fragment, derived from a sta bilized mutant of the phosphorylcholine binding antibody McPC603, was inves tigated by H/D exchange and ESI-MS and compared with the folding kinetics o f its constituting domains V-H and V-L Both V-H and V-L adopt essentially n ative-like exchange protection within the dead time of the manual-mixing H/ D exchange experiment (10 s) and in the case of V-L, which contains two cis -prolines in the native conformation, this fast protection is independent o f proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V-L and V-H. Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and shore-term denat uration. For the equilibrium-denatured scFv fragment, whose native structur e formation is dependent on a cis conformation of an interface proline in V L, this cis/trans isomerization reaction proceeds about one order in magnit ude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in ter ms of ii general kinetic scheme involving intermediates with and without do main association.