Comparison of the amide proton exchange behavior of the rapidly formed folding intermediate and the native state of an antibody scFv fragment.

We have investigated the stability of backbone amide protons of the intermediate and the native state of the scFv fragment of an antibody. Stopped flow experiments analyzed by MS and NMR detected the formation of an exchange protected intermediate within the deadtime of the stopped flow apparatus (17 ms). H/D exchange rates of the native protein identified a number of very stable backbone amide protons in the V(L) and the V(H) domains. In the V(L) domain, this slowly exchanging core of the scFv fragment is similar to the folding core of the intermediate, while the V(H) domain possesses a great number of very stable amide protons which are not stabilized to a significant degree in the folding intermediate of the scFv fragment.