The rate-limiting steps for the folding of an antibody scFv fragment

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl-peptide bonds which are cis in the native state affect the refolding kinetics of long-term denatured protein. The rate-limiting step is the trans --> cis isomerization at the ProL95-peptide bond, which is catalyzed by peptidyl-prolyl-cis/trans-isomerase (PPIase), and is the prerequisite for correct V(H)/V(L) domain association. Refolding of short-term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis --> trans re-isomerization at the ProL95-peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short-term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate-limiting.