Abstract
Nanobodies (NB) are powerful tools for biotechnological and therapeutic applications. They strongly bind to their targets and are very stable. Early studies showed that NB unfolding is reversible and can be analyzed by equilibrium thermodynamics, whereas more recent studies focused on their kinetic stability in very harsh conditions that are far from storage or physiological temperatures (4-37 °C). Here, we show that the thermodynamic view of NB stability holds in a wide range of temperatures (18-100 °C). The thermodynamic stability of three different NBs did not correlate with binding affinity for their target. Alpha-Fold 2 analyses of these NBs showed structural differences in the binding site and hydrogen bond networks. We expect that our approach will be helpful to improve our capacity to enhance structure-function-stability relationships of NB.