Abstract：
The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total β-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.
摘要：
这项工作介绍了假结核耶尔森氏菌（YpOmpF）的非特异性OmpF孔蛋白在苛刻的变性条件下形成淀粉样蛋白样结构的体外和计算机研究结果，具有β桶构象的膜蛋白。已经表明，为了获得淀粉样蛋白样孔蛋白聚集体，必须在高温下在酸性pH的缓冲溶液中使其结构初步不稳定，然后在室温下长期孵育。在pH4.5的溶液中于95°C加热后，在蛋白质的三级和二级结构水平的孔蛋白分子中观察到明显的构象重排，伴随着总β结构含量的增加和蛋白质溶液特征粘度值的急剧下降。随后在室温下长期暴露产生的不稳定中间体YpOmpF导致形成各种形状和大小的孔蛋白聚集体，这些聚集体结合硫黄素T，一种用于检测淀粉样蛋白结构的特定荧光染料。与最初的蛋白质相比，淀粉样蛋白途径的早期中间体，低聚物,已显示对Neuro-2aCCL-131™小鼠神经母细胞瘤细胞具有增加的毒性。对蛋白质聚集过程中固有荧光变化的计算机建模和分析结果表明，在淀粉样聚集体形成过程中，YpOmpF结构的变化不仅影响与孔的外环相对应的内部无序结构的区域，但也是分子的主要框架，具有β桶固有的刚性空间结构。
