Formation of Amyloid-Like Conformational States of ß-Structured Membrane Proteins on the Example of OMPF Porin from the Yersinia pseudotuberculosis Outer Membrane.

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 ß-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 ß-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 ß-barrel.

Effect of Non-Specific Porins from the Outer Membrane of Yersinia pseudotuberculosis on Mice Brain Cortex Tissues.

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.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Modified and Mutant Porins in the Study on Molecular Basis of Non- Specific Diffusion.

Site-directed mutagenesis allows elucidation of the basic principles of the porin-driven membrane permeability and opens the possibility for the modulation of functional states of porin channels. The review is aimed to show the advantages of using mutant and chemically modified porins for obtaining detailed information about molecular mechanisms that underlie the non-specific transmembrane diffusion. We summarized data regarding the effects of the point substitutions and the external loop deletions on electrophysiological properties of general porins. The influence of charges inside the pore eyelet and the roles of external loops in ion conductance, ion selectivity, and voltage gating were described.

Peculiarities of thermal denaturation of OmpF porin from Yersinia ruckeri.

Irreversible denaturation of membrane proteins in detergent solutions is similar to unfolding of water-soluble multidomain proteins and represents a complex, multistage process. Pore-forming proteins of Gram-negative bacteria are heat-modifiable proteins, i.e., proteins altering their molecular forms (trimers or monomers), and accordingly, their electrophoretic mobilities depending upon denaturation conditions. There are still some contradictory data on the peculiarities of the conformational changes in the porin structure with temperature. Some authors demonstrated the loss of the porin trimeric structure only after unfolding of monomer subunits. Other researchers initially observed the dissociation of porin oligomers into the folded monomers. Using SDS-PAGE, spectroscopic methods and differential scanning calorimetry, a detailed study of thermally induced changes in the spatial structure of OmpF porin from the fish pathogen Yersinia ruckeri (Yr-OmpF) was carried out. The data obtained allowed us to conclude unambiguously that changes in the spatial structure of the monomers of Yr-OmpF precede the dissociation of the porin trimer.