That this local conformational difference in a region of the molecule which does not directly contact the can have an effect on antigen binding has been suggested in a paper by Poljak, who showed a correlation between backbone conformation, Sequence in pos.H9 and the take-off angle of CDR2 but failed to see the correation between backbone conformation and H6. However, there is a lot of anectotal evidence that frequently the substitution of H6 Gln by Glu or vice-versa induced by improper primer usage during the cloning of a vh domain can severly affect the antigen affinity.