The lack of trade-off between conformational stability and binding affinity in a nanobody with therapeutic potential for a misfolding disease.

To improve protein pharmaceuticals, we need to balance protein stability and binding affinity with in vivo efficiency. We have recently developed a nanobody (NB-AGT-2) against the alanine:glyoxylate aminotransferase with high stability (T ∼ 86 °C) that may be useful to treat a misfolding disease called primary hyperoxaluria type 1. In this work, we characterize the relationships between protein stability and binding affinity in NB-AGT-2 by generating single and double cavity-creating mutants in its hydrophobic core. These mutations decrease thermal stability by 10-20 °C, reflecting changes in thermodynamic stability of up to 8 kcal·mol, hardly affecting their binding affinity for its target. Statistical mechanical analysis support long-range propagation of stability effects due to mutations. Our results thus show that NB stability can be largely challenged without an effect on its binding.