Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas, Quito, Ecuador.
Protein Sci. 2024 Sep;33(9):e5134. doi: 10.1002/pro.5134.
Function and structure are strongly coupled in obligated oligomers such as Triosephosphate isomerase (TIM). In animals and fungi, TIM monomers are inactive and unstable. Previously, we used ancestral sequence reconstruction to study TIM evolution and found that before these lineages diverged, the last opisthokonta common ancestor of TIM (LOCATIM) was an obligated oligomer that resembles those of extant TIMs. Notably, calorimetric evidence indicated that ancestral TIM monomers are more structured than extant ones. To further increase confidence about the function, structure, and stability of the LOCATIM, in this work, we applied two different inference methodologies and the worst plausible case scenario for both of them, to infer four sequences of this ancestor and test the robustness of their physicochemical properties. The extensive biophysical characterization of the four reconstructed sequences of LOCATIM showed very similar hydrodynamic and spectroscopic properties, as well as ligand-binding energetics and catalytic parameters. Their 3D structures were also conserved. Although differences were observed in melting temperature, all LOCATIMs showed reversible urea-induced unfolding transitions, and for those that reached equilibrium, high conformational stability was estimated (ΔG = 40.6-46.2 kcal/mol). The stability of the inactive monomeric intermediates was also high (ΔG = 12.6-18.4 kcal/mol), resembling some protozoan TIMs rather than the unstable monomer observed in extant opisthokonts. A comparative analysis of the 3D structure of ancestral and extant TIMs shows a correlation between the higher stability of the ancestral monomers with the presence of several hydrogen bonds located in the "bottom" part of the barrel.
功能和结构在三磷酸甘油醛异构酶（TIM）等必需寡聚体中紧密结合。在动物和真菌中，TIM 单体是无活性和不稳定的。以前，我们使用祖先序列重建来研究 TIM 的进化，发现这些谱系分化之前，最后一个后口动物 TIM 的共同祖先（LOCATIM）是一个必需的寡聚体，类似于现存的 TIM。值得注意的是，量热法证据表明，祖先 TIM 单体比现存的单体更具结构。为了进一步提高对 LOCATIM 的功能、结构和稳定性的信心，在这项工作中，我们应用了两种不同的推断方法，并对它们进行了最坏情况的推断，以推断这个祖先的四个序列，并测试它们理化性质的稳健性。对 LOCATIM 的四个重建序列进行的广泛生物物理特性研究表明，它们具有非常相似的流体力学和光谱特性，以及配体结合的能量学和催化参数。它们的 3D 结构也保持保守。尽管在熔点上存在差异，但所有 LOCATIM 都表现出可逆的尿素诱导的展开转变，对于那些达到平衡的 LOCATIM，估计具有高构象稳定性（ΔG=40.6-46.2 kcal/mol）。无活性单体中间产物的稳定性也很高（ΔG=12.6-18.4 kcal/mol），类似于一些原生动物 TIM，而不是现存后生动物中观察到的不稳定单体。对祖先和现存 TIM 的 3D 结构的比较分析表明，祖先单体的较高稳定性与位于桶“底部”的几个氢键的存在之间存在相关性。
