FOLDING NUCLEI OF THE SCFV FRAGMENT OF AN ANTIBODY

The folding kinetics of the variable domains of the phosphorylcholine- binding antibody McPC603, combined into a scFv fragment [V-H-(Gly(4)Se r)(3)-V-L], were investigated by the use of fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). All thr ee methods gave evidence for the occurrence of a major kinetic interme diate during the refolding of the denatured, oxidized scFv fragment. T his intermediate is formed within the first 30 s of folding and compri ses exchange-protected amide protons of hydrophobic and aromatic amino acids, most of which are localized within the inner beta-sheet of the V-L domain. In the subsequent slow step, most of the amide protons be come protected with rate constants that are very similar for residues of both domains. These data are in agreement with the MS results, whic h indicate a cooperative folding event from the intermediate to the na tive state of the scFv fragment.