Occasionally, indirect experimental evidence suggests unusual structural solutions: One this slide there is a model of the scFV fragment of antibody abPC48, which lacks the conserved disulfide bridge in the VH domain (Karl Proba, Arne Wörn). One of the cysteine residues has been replaced by tyrosine. Both the disulfide-lacking scFV fragment and a disulfide restored construct were fully functional, although refolding yields for the disulfide restored construct were very poor. Refolding in the presence of a glutathione redox shuffle produced a variant in which the remaining unpaired cys was quantitatively modified by glutathion. Refolded in the absence of glutathione, the free cysteine reacted over time with the protease inhibitor PVblock, indicating thatthe residue must be accessible. The modified molecules were fully functional, indicating that the local conformation of the free cysteine had to be changed in such a way that the residue is accessible, without disturbing the antigen binding site, particularely nearby CDR H1.