An unusual cysteine V87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V22 and V92 in the variable heavy chain (V) and V23, V87 and V88 in the variable light chain (V). The influence of two unusual contiguous Cys at positions V87 and V88 was studied by considering the wild type fragment and mutant variants: V-C88S, V-C87S, V-C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V-C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V-C87 by a usual residue at this position like Tyr caused distant structural changes at the V region that confers a higher mobility to the V-CDR2 and V-CDR3 loops improving the scFv binding to the antigen.