A calorimetric and structural analysis of cooperativity in the thermal unfolding of the PDZ tandem of human Syntenin-1 Martínez Herrerías, José Cristóbal Syntenin-1 is a multidomain protein containing a central tandem of two PDZ domains flanked by two unnamed domains. Previous structural and biophysical studies show that the two PDZ domains are functional both isolated and in tandem, occurring a gain in their respective binding affinities when joined through its natural short linker. To get insight into the molecular and energetic reasons of such a gain, here, the first thermodynamic charac terization of the conformational equilibrium of Syntenin-1 is presented, with special focus on its PDZ domains. These studies include the thermal unfolding of the whole protein, the PDZ-tandem construct and the two isolated PDZ domains using circular dichroism, differential scanning fluorimetry and differential scanning calorimetry. The isolated PDZ domains show low stability (ΔG < 10 kJ⋅mol− 1) and poor cooperativity compared to the PDZ tandem, which shows higher stability (20–30 kJ⋅mol− 1) and a fully cooperative behaviour, with energetics similar to that previously described for archetypical PDZ domains. The high-resolution structures suggest that this remarkable increase in cooperativity is associated to strong, water-mediated, interactions at the interface between the PDZ domains, associated to nine conserved hydration regions. The low Tm value (45 ◦C), the anomalously high unfolding enthalpy (>400 kJ⋅mol− 1), and native heat capacity values (above 40 kJ⋅K− 1⋅mol− 1), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics. 2023-05-15T11:44:09Z 2023-05-15T11:44:09Z 2023-04-24 info:eu-repo/semantics/article Martinez JC, Ruiz-Sanz J, Resina MJ, Montero F, Camara-Artigas A, Luque I (2023) A calorimetric and structural analysis of cooperativity in the thermal unfolding of the PDZ tandem of human Syntenin-1. Int. J. Biol. Macromol. 242, 124662 https://hdl.handle.net/10481/81544 https://doi.org/10.1016/j.ijbiomac.2023.124662 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier