Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding

While studying the expression of single-chain antibodies (scFv) derived fro m several murine monoclonal antibodies, we found that residue 6 in Framewor k region 1 of the heavy chain variable domain plays a crucial role in antib ody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this positio n the mild-type residue glutamine (Q) was substituted by glutamate (E), Inc reased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutant s. Grafting of the three additional class IA derived FR1 residues, based up on the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of t he Q6E-containing scFv, Our results suggest that residue 6 of the heavy cha in may be part of a folding nucleus, involving the first two P-strands of F ramework region 1, The evolutionary conservation of either glutamine or glu tamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.