*4.4. SDS-PAGE*

Analysis of protein fractions obtained was carried out using SDS-PAGE according to the method described in [49,50]. SDS-PAGE procedure was carried out under native (solubilization temperature 25 ◦C) and denaturing (after heating at 99 ◦C) conditions. Apparent MW of proteins in different fraction was determined according to relative mobilities of standard proteins using marker proteins (Thermo Fisher Scientific, Waltham, MA, USA) with MW from 10 to 170 kDa. The proteins separated in the gel were stained with Coomassie R-250 in 3.5% perchloric acid [53]. The protein content of the samples was determined spectrophotometrically at 280 nm, assuming an optical density of 1.0 for a protein concentration of 1 mg/mL.

#### *4.5. Spectroscopic Methods*

All absorption, circular dichroism (CD), and fluorescence measurements were performed using the protein samples in buffer A, containing 0.25% SDS at 25 ◦C. UV absorption spectra were recorded on a UV-visible spectrophotometer UV-1601PC (Shimadzu, Japan) in quartz cuvettes with the path-length of 1 cm. The correction for light scattering of the protein solution was carried out as described in [54]. Specific absorption factor A 0.1%/1 cm of porin was taken equal to 1.00.

*Circular dichroism (CD) spectra* were recorded on Chirascan Plus CD spectropolarimeter (Applied Photophysics Limited, Leatherhead, UK) in quartz cuvettes with the optical path length of 0.1 and 1 cm for the far-UV or peptide (180–250 nm) and the near-UV or aromatic (250–350 nm) regions, respectively. Samples with the protein concentrations of 1.0 μM and 6.0 μM were used for the CD measurements in the peptide and aromatic regions, respectively.

In the aromatic spectral region, ellipticity [θ]M was calculated as molar ellipticity taking the molecular weights of the protein monomer and trimer as 35 and 105 kDa, respectively. In the peptide region, ellipticity was calculated as the mean residue ellipticity taking the molecular weight of amino acid residue as 110 Da, in standard units (deg cm2 dmol<sup>−</sup>1) by the equation:

$$\begin{bmatrix} \{\theta\} \end{bmatrix} = \begin{bmatrix} \{\theta\}\_{\text{obs}} \cdot \text{S} \cdot \text{110/10} \cdot \text{C} \cdot \text{l} \end{bmatrix} \tag{1}$$

where S is the sensitivity; C is the protein concentration, mg/mL; and l is the optical path length, dm.

The content of the secondary structure elements of the protein was calculated with CD Pro software [34].

*Intrinsic protein fluorescence spectra* of the porin were measured in quartz cuvettes with optical path length of 1 cm using a RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). Porin samples with protein concentration of 0.5 μM were prepared in buffer A, containing 0.25% SDS. Fluorescence was excited by the light with wavelength of 280, 296, and 305 nm. The excitation and emission slits were set at 5 nm. Fluorescence spectra corrected by rhodamine B (Wako Pure Chemical Industries, Osaka, Japan) were recorded by subtracking the Raman band of the buffer solution.

### *4.6. Lipid Bilayer Experiments*

The experiments on reconstitution of protein samples into the BLM were performed under the symmetric conditions, so that the investigated proteins were present on both sides of the BLM. Fluctuations of the current through the BLM in the presence of the porin were measured in the clamp mode on the membrane potential. The current from the membrane was conducted by Ag/AgCl electrodes and registered at a membrane potential of 10–50 mV. The electrical parameters of the BLM were measured with bilayer clamp amplifier BC-535, pCLAMP 10 and Clampfit 10 software suites.

The BLM was formed from a 1% solution of 1-monooleoylglycerol (monoolein) (Sigma- Aldrich, St. Louis, MO, USA) in *n*-heptane with a pipette (a hole of 0.25 mm in diameter) on a Teflon cup placed in a thermostated optically transparent cuvette filled with an electrolyte. The experiments of protein reconstitution in the BLM were performed at room temperature (22 ◦C). The aqueous phase contained 0.2–2 M NaCl in buffer A and the studied protein in the range of concentrations of 10–100 ng/mL.

#### *4.7. Amino Acid Composition and N-Terminal Sequencing*

*Amino acid analysis* was performed by using a Biochrom 30 amino acid analyzer (Biochrom Ltd. CB4 OFJ, Cambridge, UK), after hydrolysis of purified protein with 6 N HCl according to the method descried in [55]. The amino acid content of the protein sample was calculated in Mol% of each amino acid.

*DNA isolation.* Preparation of genomic DNA for whole genome sequencing (WGS) was performed according to the protocols [56].

*Genome sequencing and assembly*. Two kits were used for library preparation: KAPA HyperPlus (Roche, Basel, Switzerland) and Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA). Libraries were checked on a Bioanalyzer (Agilent, Santa Clara, CA, US) and sequenced on an Illumina MiSeq instrument with paired-end protocol. Genome assembly was performed with CLC Genomic Workbench v.12.

*Genome annotation*. Genome was annotated by the Rapid Annotations using Subsystems Technology (RAST) server [57,58].

GenBank NCBI submission. The obtained *Marinomonas primoryensis* KMM 3633<sup>T</sup> genome sequence was submitted in NCBI SRA database under the BioProject number: PRJNA622482. DNA of Porin\_4 was translated, complementary annotated in NCBI by conserved domains detection and submitted in GenBank with Accession Number MK820371 (Protein Accession-QES04118).

Sequences, used for analysis. *Marinomonas primoryensis* strain AceL (WP\_112140846), *Marinomonas* sp. IMCC 4694 (WP\_148833815), *Polaromonas* sp. JS666 (Q123M1), *Magnetospirillum magneticum* AMB-1 (Q2WBE6), *Photobacterium damselae subsp. damselae* (KJF82573.1), *Escherichia coli* (WP\_000768382.1), *Basfia succiniciproducens* MBEL55E (Q65V42).

*Bioinformatic analysis*. Alignment of protein sequences was performed by NCBI BLAST, signaling peptide was predicted by Phobius server (http://phobius.sbc.su.se/) [59]. ProtParam tool was used for the computation of various physical and chemical parameters for the proteins (https://web.expasy.org/ protparam/) [60].

Polarity index was calculated according to following formula [30]:

(Asx + Glx + Lys + Arg + Ser + Thr)/(Val + Leu + Ile + Met + Pro + Phe). For the polarity calculation was used formula recommended by [29]:

(Asx + Glx + Lys + Arg) + (Ser + Thr + Tyr + His + Gly)/2

PSORTb program was used for bacterial protein subcellular localization prediction (https://www. psort.org/psortb/) [61,62].

Pfam server was used for protein classification (http://pfam.xfam.org/) [63].

TCBD (Transporter Classification DataBase) and bioinformatics resources of this base were used for porin classification [63].

**Author Contributions:** O.D.N. Conceptualization, project administration, literature analysis, data analysis and interpretation, writing and editing of the manuscript. V.A.K. Performed experiments (*M. primoryensis KMM* 3633<sup>T</sup> porin isolation), data analysis and interpretation. N.Y.K. Performed experiments (optical properties of *M. primoryensis KMM* 3633<sup>T</sup> porin), data analysis. G.N.L. Performed BLM experiments and interpretation of data obtained. L.A.R. isolation and identification of novel *Marinomonas* strain. Cultivation of the microbial cells. O.Y.P. Data analysis and interpretation, writing original draft. T.F.S. Literature analysis, discussion of manuscript structure. E.I.A., M.S.K., N.N.R. and O.L.V. contributed in genome sequencing, assembling, annotation and analysis, in comparison of *M. primoryensis KMM* 3633<sup>T</sup> porin with porins in different Data Bases, analysis of amino acides composition and theoretical porin properties. O.L.V. contributed in literature analysis, discussion of manuscript structure and in manuscript writing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Russian Foundation for Basic Research grant № 19-04-00318.

**Acknowledgments:** The authors of the article are grateful to Victor Emelyanenko (Institute for Biological Instrumentation, Institute of Theoretical and Experimental Biophysics, Pushchino, Russia) for a helpful discussion.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
