The invariant tryptophan in an H chain V region is not essential to antibody binding.

The first amino acid residue of the second framework region in all antibody H and L chain V regions sequenced to date is invariably tryptophan. To test whether this invariance is essential to proper domain folding and generation of a functional antibody, the tryptophan residue in the heavy chain V region of a mouse anti-p-azophenylarsonate antibody was converted to an alanine residue by oligonucleotide-directed mutagenesis of the H chain gene. The mutant gene was transfected into mouse hybridoma cells that produce the homologous L chain, and the resulting mutant antibody was purified from the cell supernatant. It was shown to have essentially the same reactivity as wild type toward a series of anti-idiotypic antibodies and to bind Ag with a Ka similar to that of wild type.