*2.5. CD Spectroscopy*

The secondary structure of the recombinant proteins was determined by CD spectroscopy in the far ultraviolet region. CD spectra of PldA-GFP, PldA and GFP in 0.03% SDS (exposure time from 10 min to 2 h and 24 h) and in 0.1% SDS (incubation time 2 and 24 h), as well as the spectrum of GFP in PBS were obtained (Figure 8A). According to turbidimetry data (Figure 6A), in these solutions of the detergent, almost complete solubilization of the recombinant protein from IBs is observed.

**Figure 8.** Far UV CD spectra of PldA-GFP, PldA and GFP. (**A**) PldA-GFP (•), GFP (Δ), and PldA (-): 0.03% SDS, 10 min; PldA-GFP () and GFP (): 0.1% SDS, 2 h; GFP: PBS (-); (**B**) PldA-GFP: 4 M urea, 2 h (•) and 24 h (-).

Recombinant GFP in PBS has a CD spectrum with a maximum at 198 nm and only one minimum at 220 nm, characteristic of proteins with β-pleated sheet structure (Figure 8A). As shown earlier [30], the *Y. pseudotuberculosis* phospholipase A1, which, similar to GFP, has the cylindrical β-sheet structure, exhibits a CD spectrum with a positive band at 193 nm and a broad negative band centered at 215 nm. The CD spectra of the recombinant proteins PldA-GFP, PldA and GFP in SDS solutions are characterized by a maximum at 197 nm and two minima at 208–207 nm and 219–217 nm, and are typical for mixed α-β proteins. Increasing the SDS concentration and incubation time caused an increase in the spectra amplitude and a decrease in the peak ratio at 218 and 207 nm which indicates an increase in the content of the α-helix in the protein (Figure 8A).

Using the CDPro software [31] the content of secondary structure elements of the recombinant proteins was determined (Table 1).

**Table 1.** Content of secondary structure elements (%) in PldA-GFP, PldA and GFP, dissolved in SDS and PBS.


\* UniProtKB—POA921 (PA1-*E.coli*).

As can be seen from Table 1, in 0.03% SDS PldA-GFP, PldA, and GFP have a pronounced secondary structure including α-helices and β-pleated sheets. However, the content of α-helices and random coil structure in these recombinant proteins is higher than in the corresponding native proteins. These data suggest that in the studied proteins, molecules with a native-like structure, which are fluorescently active, coexist with partially and misfolded polypeptides or segments. An increase in SDS concentration and incubation time in detergent solutions leads to an increase in the content of α-helices and a decrease in the content of β-structure. At the same time, the content of regular (α-helix and β-sheet) and random coil structure remains practically unchanged, and is about 50% and 28–32%, respectively. The data obtained suggest that SDS induces the β-sheet to α-helix structural conversion in the recombinant proteins. A similar type of denaturation by SDS has been found for some globular proteins that have a β-sheet structure [34]. In addition, it has been shown earlier that SDS-denatured PldA *E. coli* has a non-native secondary structure with a high α-helix content [35].

As follows from the data presented in Table 1, PldA-GFP and PldA, dissolved from IBs with SDS at the same conditions, differ significantly in the content of β-structure, which is 3–4 times lower in PldA than in the fusion protein. Based on these data, it can be assumed that PldA-moiety in the chimeric protein, as compared to PldA, has a secondary structure that is closer to the native one, and is more resistant to denaturation by SDS. Thus, the use of GFP as a fusion partner appears to improve the folding of PldA when expressed in *E. coli.*

Comparative analysis of the spectral data of GFP and PldA-GFP solubilized in 0.1% SDS during incubation for 2 and 24 h showed that the structural changes of GFP induced by SDS, compared with those of PldA-GFP, occur more slowly and lead to a more significant decrease in the content of β-structure in the protein (Figure 8, Table 1). As a result, we can assume that GFP is more sensitive to SDS-denaturation than GFP-moiety in chimeric protein.

The secondary structure of PldA-GFP solubilized from IBs with 4 M urea was also determined. The CD spectrum of the recombinant protein in urea (incubation time 2 h) has a maximum at 197 nm, minima at 225 and 218 nm, and is specific for proteins with a β-structure (Figure 8B). The long-wavelength position of the negative band (225 nm) in the CD spectrum is probably due to the aggregated state of the solubilized protein. An increase in exposure time of the IBs in urea solution to 24 h leads to the widening of the PldA-GFP spectrum with a decrease in the ellipticity of the positive and negative (−5294 to −3220 deg cm2 dmol−1) bands, a shift in the positions of the minima to 217 and 211 nm, and the appearance of an intense negative band at 193 nm, which indicates an increase in the content of random coil structures in the protein. A quantitative analysis of CD spectra of PldA-GFP indicates a decrease in the content of both α-helix (8.8 to 0.6%) and β-sheet (34.7 to 31.7%) with an increase in random coil structure fraction (33.8 to 45.5%), when the incubation time of protein in urea increases. We can assume that urea not only dissolves the recombinant protein from IBs, but also denatures it. It should be noted that denaturation of *E. coli* PldA in the presence of urea is accompanied by the formation of a random coil structure [35]. Thus, urea induces an increase in the content of random coil conformation in PldA-GFP, while maintaining a high percentage of β-sheet structure. The protein species with a native-like β-sheet structure appear to be more resistance to urea, than misfolded or partially folded polypeptides.
