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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.

It was found that a single-dose immunization of mice with Yersinia pseudotuberculosis porins OmpF and OmpC causes development of pathological changes in the deep layers of cerebral cortex characterized by dystrophic changes in the cells against the background of the increasing titer of specific antibodies. At the same time, the increased level of caspase-3 expression is observed in the neurons, which indicates induction of proapoptotic signaling pathways. The obtained results indicate potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate development of degenerative changes in brain cells.

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins., (Copyright © 2022. Published by Elsevier B.V.)

The main limitation for practical implementation of quantum dots-based sensors and biosensors is the possible contamination of sensing media with quantum dots (QDs) moved out from the sensor structure, being critical for living systems measurements. Numerous efforts have addressed the challenge of pre-synthesized QDs incorporation into porous matrix provide, on the one hand, proper fixation of quantum dots in its volume and preserving a free analyte transfer from the sensing media to them - on the other hand. Here, we propose an alternative insight into this problem. Instead of using preliminary synthesized particles for doping a matrix, we have in situ synthesized cadmium sulfide QDs in porous biopolymeric matrices, both in an aqueous solution and on a mica substrate. The proposed technique allows obtaining QDs in a matrix acting simultaneously as a ligand passivating surface defects and preventing QDs aggregation. The conjugates were used as a photoluminescence sensor for the metal ions and glutathione detection in an aqueous media. Different kinds of sensor responses have been found depending on the analyte nature. Zinc ions' presence initiates the intraband QDs emission increases due to the reduction of non-radiative processes. The presence of copper ions, in contrast, leads to a gradual photoluminescence decrease due to the formation of the non-luminescent copper-based alloy in the QDs structure. Finally, the presence of glutathione initiates a ligand exchange process followed by some QDs surface treatment enhancing defect-related photoluminescence. As a result, three different kinds of sensor responses for three analytes allow claiming development of a new selective QD-based sensor suitable for biomedical applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)