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 in termediate and the native state of the scFv fragment of an antibody, S topped flow experiments analyzed by MS and NMR detected the formation of an exchange protected intermediate within the deadtime of the stopp ed flow apparatus (17 ms), H/D exchange rates of the native protein id entified 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 th e scFv fragment is similar to the folding core of the intermediate, wh ile the V-H domain possesses a great number of very stable amide proto ns which are not stabilized to a significant degree in the folding int ermediate of the scFv fragment. (C) 1997 Federation of European Bioche mical Societies.