Department of Physical Chemistry, Institute of Biotechnology and Excellence Unit in Chemistry Applied to Biomedicine and Environment, Faculty of Sciences, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain.
Department of Chemistry and Physics, Agrifood Campus of International Excellence (ceiA3) and CIAMBITAL, University of Almería, Carretera de Sacramento s/n, 04120 Almería, Spain.
Int J Biol Macromol. 2023 Jul 1;242(Pt 1):124662. doi: 10.1016/j.ijbiomac.2023.124662. Epub 2023 Apr 27.
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 characterization 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) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20-30 kJ·mol) 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 T value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol), and native heat capacity values (above 40 kJ·K·mol), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics.
衔接蛋白-1 是一种含有中央 PDZ 结构域串联的多功能蛋白，两侧为两个未命名的结构域。先前的结构和生物物理研究表明，两个 PDZ 结构域在单独和串联时都具有功能，当通过其自然短接头连接时，它们各自的结合亲和力会增加。为了深入了解这种增益的分子和能量原因，本文首次对衔接蛋白-1 的构象平衡进行了热力学特征描述，特别关注其 PDZ 结构域。这些研究包括使用圆二色性、差示扫描荧光法和差示扫描量热法对整个蛋白质、PDZ-串联结构和两个分离的 PDZ 结构域进行热变性。与 PDZ-串联相比，分离的 PDZ 结构域的稳定性较低（ΔG < 10 kJ·mol），协同性较差，而 PDZ-串联则具有更高的稳定性（20-30 kJ·mol）和完全协同性，其热力学性质与先前描述的典型 PDZ 结构域相似。高分辨率结构表明，这种协同性的显著增加与 PDZ 结构域之间界面处的强、水介导的相互作用有关，与九个保守的水合区域有关。低 T 值（45°C）、异常高的变性焓（>400 kJ·mol）和天然热容值（高于 40 kJ·K·mol）表明，这些界面埋藏水在衔接蛋白-1 折叠能量学中起着重要作用。
