Stability and plasticity play important roles in the affinity maturation of anti-protein antibodies

CAUERFF, ANA

Rosario, Santa Fé, Argentina

Simposio; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The biotechnological and industrial applications of proteins are usually limited by deficient protein stability. The objective of this work is to study the structural basis of the affinity maturation of two anti-lysozyme (HEL) antibodies, in relationship with the stability of their variable domains. These two mAbs, F10.6.6 and D44.1, are derived from the same germlines genes, and recognize the same epitope on HEL surface, but F10.6.6 has ~103 higher affinity towards HEL than D44.1.To study the contribution of each variable region of both mAbs in binding kinetics, thermodynamics and stability, chain shuffling experiments were performed. It was noticeable that VH domain drives the association rates to HEL while VL domain modulates the dissociation rate and binding enthalpy. Crystallographic analysis of the free and bound form of both antibodies shows that F10.6.6 losses more contacts in VH-VL interaction than D44.1 upon binding to HEL. Chemical and thermal stability was studied by circular dichroism and intrinsic fluorescence measurements. Fv F10.6.6 has an increased thermal and chemical stability compared with Fv D44.1, showing a cooperative unfolding transition. As a conclusion, an improvement of the stability of the variable domain that increases the plasticity of the VH-VL interaction results in the improvement of the binding properties of the antibody towards the antigen.