2.4.1. CD and Intrinsic Protein Fluorescence Spectra

One of the important physicochemical characteristics of proteins is their resistance to various denaturing agents and temperature. Therefore, we paid special attention to conformational changes in MpOmp molecule under various conditions: (1) upon dilution of the protein solution, (2) in the presence of non-ionic (POE) and ionic (SDS) detergents, and (3) upon heating. For this purpose, CD analysis and intrinsic protein fluorescence were used. CD spectra were recorded in the far and near UV regions. Intrinsic protein fluorescence spectra were recorded at an excitation wavelength of 280 nm.

It was found that conformational changes were not observed either at the level of the secondary or at the level of the tertiary structure of MpOmp upon dilution of the protein solution without any detergents (Figure 3A,B).

**Figure 3.** CD spectra in peptide regions (**A**,**C**,**E**) and intrinsic fluorescence spectra atλex 280 nm (**B**,**D**,**F**) of *M. primoryensis* porin (MpOmp) in Tris-HCl buffer(**A**,**B**) and in the presence of POE (**C**,**D**) and SDS (**E**,**F**). The protein samples of MpOmp (1.0 μM and 6.0 μM for peptide and aromatic regions, respectively) were dissolved in 10 mM Tris-HCl buffer, pH 7.5, containing 0.5% POE (1) or 0.25% SDS (2). Samples 2, 3, 4, and 5 were heated at 30, 40, 50, and 80 ◦C, respectively (**C**,**D**,**E**,**F**).

CD spectra were recorded in the far and near UV regions. CD spectra of MpOmp in the aromatic region (240–320 nm) have a low amplitude and low resolution, which indicates a loosened tertiary structure because of the apparently weak interaction between the protein monomers (data not shown).

CD spectra of the porin studied between 180–240 nm (the region of peptide bonds) in the presence of non-ionic detergent POE are characterized by positive band at 195 nm and only one negative minimum that was centered at 220 nm. The spectra crossed the zero line at 209 nm (Figure 3C). It is typical for proteins with β-pleated sheet structure of (α + β) type [32]. In the presence of SDS a new negative band at 207 nm was observed (Figure 3E), which indicated α-helical structure formation typical of denatured proteins in SDS [33].

Using the CDPro software [34] the content of secondary structure elements of MpOmp was determined. As we can see in Table 3, the secondary structure of the protein in solution of various detergents differed. In POE solution the total β-structure of MpOmp accounted 70% while alpha α-helices make up only 5%. Such ratio of the elements of the regular secondary structure of the protein is similar to that of the porins in the native environment in the bacterial membrane [35].


**Table 3.** Content of secondary structural elements in porin from *M. primoryensis* KMM 3633<sup>T</sup> (%).

However, in the presence of SDS, the polypeptide chain of the studied protein contains a significant amount of α-helical regions (3.6 times more than in a solution of non-ionic detergent) and a smaller amount of total β-structure. These data indicate that ionic detergent SDS has a denaturing effect on the secondary structure of MpOmp. Similar denaturing effect of this ionic detergent we observed earlier for *Yersinia* porins [36].

In Figure 3 the intrinsic fluorescence spectra of MpOmp in solutions of POE (Figure 3D) and SDS (Figure 3F) are presented. A significant shift of the maximum of the spectrum to the short- wavelength region in the presence of ionic and nonionic detergents indicates that the radiation of the protein occurs because of tyrosine residues. These results are consistent with the data of amino acid sequence (Table 2), according to which the tryptophan residues are absent in the protein studied.

Thus, according to data of optical spectroscopy the secondary structure of MpOmp depends on the detergent nature present in its solution.
