PATHWAY OF DETERGENT-MEDIATED AND PEPTIDE LIGAND-MEDIATED REFOLDING OF HETERODIMERIC CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) MOLECULES

We investigated the mechanism of refolding and reassembly of recombina nt alpha and beta chains of the class II major histocompatibility mole cules (MHC-II) HLA-DRB50101. Both chains were expressed in the cytoso l of Escherichia coli, purified in urea and SDS, and reassembled to fu nctional heterodimers by relacement of SDS by mild detergents, incubat ion in a redox-shuffling buffer and finally by oxidation and removal o f detergent, Refolding was mediated by mild detergents and by peptide ligands. Early stages, of structure formation were characterized by ci rcular dichroism, fluorescence, and time-resolved fluorescence anisotr opy decay (FAD) spectroscopies. We found that formation of secondary s tructure was delectable after replacement of SDS by mild detergents. A t that stage the alpha and beta chains were still monomeric. the buffe r was strongly reducing, and the folding intermediates did not yet int eract with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild deter gents. We conclude that at that stage a tertiary structure close to th e native structure is formed at least locally. The nature and concentr ation of detergent critically determined the refolding efficiency, We compared detergents with different carbohydrate headgroups, and with a liphatic chains ranging from C-6 to C-14 in length, For each of the de tergents we observed a narrow concentration range fur mediating refold ing. Surprisingly, detergents with long aliphatic chains hail to be us ed at higher concentrations than short-chain detergents, indicating th at increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structu re formation and interactions of detergents with stable folding interm ediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.