Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain V(H)H antibodies

The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Single-domain V(H)H antibodies exhibit high specificity and affinity but have higher stability and solubility than conventional antibodies as they are single-domain proteins. In this work, based on physicochemical measurements and molecular dynamics (MD) simulations, we have gained insight that will facilitate rational design of single-chain V(H)H antibodies. We first assessed two homologous V(H)H antibodies by differential scanning calorimetry (DSC); one had a high (64.8 °C) and the other a low (58.6 °C) melting temperature. We then generated a series of the variants of the low stability antibody and analyzed their thermal stabilities by DSC and characterized their structures through MD simulations. We found that a single mutation that resulted in 8.2 °C improvement in melting temperature resulted in binding affinity an order of magnitude lower than the parent antibody, likely due to a shift of conformational space explored by the single-chain V(H)H antibody. These results suggest that the delicate balance among conformational stability, binding capability, and conformational space explored by antibodies must be considered in design of fully functional single-chain V(H)H antibodies.