*3.3. Physico-Chemical Properties of Porin from M. primoryensis KMM 3633<sup>T</sup>*

The amino acid composition of porin-like protein from *M. primoryensis* KMM 3633T has the same characteristic features as those of the classical gram-negative porins. For example, the protein composition is distinguished by a high content of acidic amino acids and low content of sulfur-containing amino acids. Cysteine is completely absent. However, there is a slight difference in the content of individual amino acids in composition of the protein characterized: it contains 1.4 times less aromatic acids and almost 1.4 times less tyrosine residues compared to the OmpF porin *E. coli.* In addition, the composition and properties of MpOmp are quite remarkable, primarily because of the fact that, in contrast to the porins of the other Proteobacteria under consideration (except for the porins from bacteria of the genus *Marinomonas* and *Photobacterium damselae subsp. damselae* presented in Table 1), there are no tryptophan residues in its molecule.

Considering the monomer stability of the analyzed porins, we noticed that predicted instability index had the lowest value for porins of *M. primoryensis* KMM 3633<sup>T</sup> and *M. primoryensis* AceL. When characterizing the monomers of these porins as the most stable, we take into account that in the bacterial cell the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu, as noted Gamage, et al. in their study [43]. In connection with the foregoing, we should especially dwell on the instability of MpOmp oligomeric structure that we observe.

Porin *M. primoryensis* KMM 3633<sup>T</sup> was isolated in the oligomeric form, however porin trimers were extremely unstable. The dissociation of MpOmp trimer into monomers, accompanied by an irreversible conformational transition was already observed at 30 ◦C. In a number of cases, on the electropherogram of the fraction of OM proteins obtained under denaturing conditions of SDS-PAGE (0.1% SDS), the protein monomer of MpOmp appeared without prior boiling of the sample. In addition, MpOmp trimer dissociated into monomers also after protein precipitation with ethanol in the presence of EDTA, the method used for purification of membrane proteins [25]. So, a trimeric form of the protein was clearly observed only under mild condition of solubilization. Therefore we cannot exclude that isolation conditions of target protein chosen by us do not lead to a partial dissociation of its trimeric form during obtaining of cell envelope and purification from cytoplasmic proteins. Thus, in order to obtain and preserve OM protein from *M. primoryensis* KMM 3633<sup>T</sup> as a trimer with a relatively stable structure, it is necessary to choose only a mild non-ionic detergent and strictly monitor the temperature at which the experiment is conducted. Similar instability of native subunit structure of some bacterial

pore-forming proteins was described in literature [44,45]. Authors of these articles should have used cross-linking in order to reveal the native oligomers of the proteins studied.

Elucidation of the conformational stability of the porin studied under various conditions using the methods of optical spectroscopy and electrophoresis made it possible to draw the following conclusion. It was found that no conformational changes were observed either at the level of the secondary or at the level of the tertiary structure of the protein upon dilution. However, the presence of ionic and nonionic detergents influenced the spatial organization of MpOmp differently and introduced characteristic features into the conformation of the protein molecule. It has been shown that the SDS ionic detergent has a denaturing effect on the conformation of the porin trimer from *M. primoryensis* KMM 3633T at the secondary protein structure, significantly increasing the content of the α-helix and decreasing the content of the total β-structure. This is consistent with the widespread literature hypothesis that SDS micelles promote the formation of α-helices in the case of β-structured proteins, containing intrinsically disordered regions (IDRs) [46]. In contrast, in the presence of a non-ionic detergent, minimal changes in protein structure were observed. Thus, the data obtained show, that non-ionic detergent POE is most suitable for solubilization of MpOmp, since in its presence the conformation of the studied protein is as close as possible to the native one.

According to modern concepts, porins are unusual membrane proteins containing a significant amount of hydrophilic amino acid residues. They are able to form β-barrels in contrast to the α-helices of almost all other membrane proteins. The association of monomers within native trimer is stabilized through the hydrophobic and hydrophilic interaction at the subunit interface and involves 35% of the barrel surface area [47].

In addition, porins are amyloidogenic proteins capable of forming amyloid-like structures under certain conditions. This may be facilitated by the availability of hydrophobic sites on the surface of the monomer, buried in the trimer, but released during its dissociation. At the same time, according to the recently published theory of the formation of multimeric proteins, the formation of their quaternary structure occurs because of the balance between the energy of intramolecular interaction and the energy of adhesion between subunits [48].

Given these facts, it can be assumed that the instability of the trimeric form of MpOmp can be caused both by the difference in the values of the indicated energies in favor of the predominant existence of a protein in the form of a monomer, and by differences in the primary structure of porins from marine and terrestrial bacteria, the trimer form of the last exhibit the high thermal stability. So, for example, mutation of residues involved in ionic interactions between the two subunits have been shown to reduce the thermal stability of the trimer significantly [49].

At present, computer models are widely used in investigation of the stabilizing forces in quaternary structure formation. Currently, there is the possibility of a detailed study of protein–protein interaction in the formation of the oligomeric structure of beta-barrel proteins [50]. Transmembrane domains of β-barrel membrane proteins have shown the presence of so called "weakly stable regions" despite an extensive network of hydrogen bonds, as well as ionic and hydrophobic interactions that give high strength to the molecule as a whole [51]. In addition, it was found that one of the ways to stabilize these areas can be by interacting with surrounding lipids [52]. On the other hand, it is known that oligomerization of the proteins, including membrane proteins, can bring various functionally important advantages to a particular protein.

Taking into account the foregoing, we can assume the following. Since the MpOmp monomer has very high stability (Table 1), and the oligomeric structure of the isolated protein, on the contrary, is extremely unstable under the heating, it is possible that the existence of the MpOmp trimer is crucial for the manifestation of the functional activity of this porin in the native membrane. This assumption is based on the fact that isolated MpOmp in monomeric form had a very low efficiency of reconstitution into an artificial bilayer. Given the fact that lipid–protein interaction can be a factor stabilizing the trimeric structure of MpOmp, the study of the pore-forming activity of this novel marine porin depending on the composition of the lipid membrane will undoubtedly be of fundamental interest.
