THE NATURE OF ANTIBODY HEAVY-CHAIN RESIDUE H6 STRONGLY INFLUENCES THESTABILITY OF A V-H DOMAIN LACKING THE DISULFIDE BRIDGE

Monoclonal antibody mAb 03/01/01, directed against the musk odorant tr aseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conse rved disulfide bridge. We investigated the energetic consequence of re storing the disulfide bond and the nature of residue H6 (Glu or Gin), which is poised to interact with Ser H92 in the recombinant scFv fragm ent obtained from this antibody. In the scFv fragment derived from thi s antibody, the stabilizing effect of Gin H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have an alyzed the conformation and hydrogen bond pattern of Gin H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to V-H domain stability. We also show t hat the unpaired cysteine H22 is buried, and conforms to the expected V-H structure. The antibody appears to have acquired two somatic mutat ions (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on V-H stability. The overall domain stabili ty is the decisive factor for generating functional, disulfide-free an tibody domains, and several key residues play dominant roles. (C) 1998 Academic Press.