Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment.
The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-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 of 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 short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude 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 terms of a general kinetic scheme involving intermediates with and without domain association.
Jäger
M
M
Biochemisches Institut, Universitit Zürich, Switzerland.
Plückthun
A
A
D003903:Deuterium; D006859:Hydrogen; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D006859:Hydrogen; D011392:Proline; D003903:Deuterium