Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies
Emina Ikeuchi, Daisuke Kuroda, Makoto Nakakido, Akikazu Murakami, Kouhei Tsumoto
The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Single-domain VHH 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 VHH antibodies. We first assessed two homologous VHH 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 VHH 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 VHH antibodies.
VHH, Antibody, single domain