The influence of the buried glutamine or glutamate residue in position 6 on the structure of immunoglobulin variable domains

Immunoglobulin V-H domain frameworks can be grouped into four distinct type s, depending on the main-chain conformation of framework 1. Based on the an alysis of over 200 X-ray structures representing more than 100 non-redundan t V-H domain sequences, we have come to the conclusion that the marked stru ctural variability of the V-H framework 1 region is caused by three residue s: the buried side-chain of H6, which can be either a glutamate or a glutam ine residue, the residue in position H7, which may be proline only if H6 is glutamine, and by H9 (H10 according to a new consensus nomenclature), whic h has to be either glycine or proline if H6 is a glutamate residue. In natu ral antibodies, these three residues are encoded in combinations that are c ompatible with each other and with the rest of the structure and therefore will yield functional molecules. However, the degenerate primer mixtures co mmonly used for PCR cloning of antibody fragments can and frequently do int roduce out-of-context mutations to combinations that can lead to severe red uction of stability, production yield and antigen affinity. (C) 2001 Academ ic Press.