Elucidation of the structural mechanisms of the affinity maturation in the immune response of anti-NP antibodies.

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It is known that the average affinity of serum generally increases with time after immunization. This phenomenon is called affinity maturation of immune response. It has been demonstrated that the variable region of the primary response anti-NP antibody, NIG9 carry none somatic mutations, and the secondary response anti-NP antibodies are somatically mutated and have a high affinity for NP.By gene analysis it has been shown that a Trp*Leu exchange at position 33 of the heavy chain is plays a crucial role in the affinity maturation of anti-NP antibodies.On the basis of the data obtained from affinity measurements and kinetic analyzes of anti-NP antibodies, the secondary response antibodies, which have a Trp*Leu exchange at position 33 of the heavy chain, show higher rate constant for association (kon). We performed the NMR analyzes for elucidating the structural basis of this type of "kinetic maturation" by using Fab analogues selectively labeled with ^<15>N.The result of the NP-AmTEMPO binding experiments indicate that H1, H3, L1, and L3 loop form the antigen binding site of both the primary response antibody, Fab (N1G9), and the secondary response antibody, Fab (B2), and the construction of the antibody combining sites ofboth Fabs were closely similar. On the basis of the comparative analyzes of the hydrogen-deuterium exchange rates and transverse relaxation rates between Fab (N1G9) and Fab (B2), we are able to conclude that a conformational flexibility exists in the antigen binding site of Fab (B2) in the absence of the hapten, which leads hapten molecule easily accessible into antigen binding site. We therefore suggest that the conformational flexibility existing in the antigen binding site of the secondary response antibodies gives net increase in bimolecular hapten-antibody association rate constant. This type of kinetic maturation mechanism is thought to be one of the most efficient affinity maturation process.