The scFv fragment of the antibody hu4D5-8:: Evidence for early premature domain interaction in refolding

Fluorescence spectroscopy and H-1/H-2-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native V-L domain and an unfolded V-H domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with H-1/H-2-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full H-1/H-2-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments. (C) 2001 Academic Press.