Formation of Amyloid-Like Conformational States of β-Structured Membrane Proteins on the Example of the OmpF Porin from the <i>Yersinia pseudotuberculosis</i> Outer Membrane

The work presents the results of an in vitro and in silico study of the formation of amyloid-like structures under harsh denaturing conditions by the nonspecific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with a 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 an acidic pH value 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 the tertiary and secondary structure of the protein, which are accompanied by an increase in the content of the total β-structure and a sharp decrease in the value of the characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to the 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 Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of changes in intrinsic fluorescence during protein aggregation suggest that during the formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only areas with an internally disordered structure corresponding to the external loops of the porin, but also the main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.