*2.3. Stability of PldA-GFP IBs*

The IBs of the chimeric protein are rather stable in aqueous suspensions. According to the dynamic light scattering (DLS) data, IBs have a monomodal size distribution with an average hydrodynamic radius (RH) of 412 nm in PBS at a concentration of 2 to 20 μg/mL (Figure 5A). However, repeated passing of the IBs suspension (20 μg/mL) through a 0.1 mm needle, followed by sonication to homogenize aggregates in PBS, leads to the appearance of a population of small particles (40–100 nm), which account for about 18% of the total volume, and a decrease in the average IBs radius (by volume) to 367 nm (Figure 5A). As was reported earlier, sonication can disturb the structure of IBs and leads to the loss of some protein from their surface and an increase in porosity [23].

**Figure 5.** Characteristic of PldA-GFP IBs. (**A**) Particle size volume distribution of IBs in PBS (20 μg/mL): IBs intact (thin line), IBs after passing through a 0.1 mm needle and sonication (thick line); (**B**) Gel filtration chromatography of PldA-GFP solubilized in 0.1% SDS, 50 mM Tris-HCl buffer, pH 8.0, on a Superdex 200 HR column.

Stability in solutions of denaturants is an important characteristic of IBs, as it is directly related to their structural organization and conformation of the protein which forms them. As is known, IBs with the high protein content in native or native-like conformation are looser and more unstable than IBs, where the protein is mainly in the form of partially or improperly folded intermediates. They are usually dissolved in mild detergents and

solutions with a low molar concentration of chaotropic agents [24]. The investigation of the stability of PldA-GFP IBs in SDS (0.01–0.1%) and urea (1–8 M) was carried out using the method of turbidimetry and GFP fluorescence.

As we can see from Figure 6A, the optical density at 350 nm of IBs solutions drops sharply with an increase in the concentration of the detergent from 0.02 to 0.03%, indicating a reduction in the number of large particles, and reaches zero at 0.05% SDS.

**Figure 6.** Solubility of PldA-GFP IBs in SDS (**A**) and urea (**B**). Dependence of turbidity signal (solid symbols) and GFP fluorescence (empty symbols) of IBs incubated in denaturant solutions for 5 min (circle), 1 h (triangle), 2 h (rhombus), and 24 h (square) on the denaturant concentration. The experiments were performed in three biological replicates. Results show the mean ± standard deviation (SD).

Simultaneously with the solubilization of IBs, their GFP fluorescence decreases. In 0.05% SDS, the fluorescence intensity of IBs is 47% of the initial value, and remains at this level for 60 min with an increase in the concentration of the detergent to 0.1%. Increasing the incubation time to 2 h leads to the GFP fluorescence of solubilized IBs falling to a low value in 0.03% and being completely absent in 0.05% SDS.

The results obtained show that the correctly folded chimeric protein, which is present in IBs, interacts with denaturant differently. One portion of the PldA-GFP fusion protein is solubilized from IBs with a simultaneous its denaturation and loss of activity, while the other portion goes into solution, retaining the native-like conformation and the fluorescent properties. This fact may be a consequence of the fluorescently active protein different localization within IBs [25] and, therefore, the availability for the detergent, as well as differences in its macromolecular and/or supramolecular structure. The latter assumption may be supported by gel chromatography data below about the structure of the fluorescent chimeric protein.

To characterize the fluorescently active protein, IBs were solubilized in 0.1% SDS and fractionated on a Superdex 200HR column. As shown in Figure 5B, fluorescent PldA-GFP was eluted from the column as three peaks, one major and two minor, corresponding to proteins with apparent molecular weights of ~600, 423 (major), and 120 kDa. As shown above, active GFP obtained by gel chromatography on Superdex 200HR is a dimer. Based on the data of gel chromatography, we can suppose that the fluorescent chimeric protein is a dimer (MW 114 kDa), which in 0.1% SDS exists mainly as an oligomer consisting of four dimers. It is possible that correctly folded PldA-GFP molecules are capable of forming more densely packed oligomeric structures in IBs and, therefore, more resistant to denaturants than the rest of the aggregate. As is known, the quaternary structure is an important factor of the conformational stability of fluorescent proteins [26].

The stability of PldA-GFP IBs in urea was also determined (Figure 6B). The turbidity of the IBs suspension decreases with an increase in the concentration of urea and the incubation time and drops to zero at a chaotropic agent concentration of 4 M. At the same time, in 4 M urea, the protein retains its fluorescence at the level of 53, 43, and 1.2% of the original value after incubation for 5 min, 2 h, and 24 h, respectively. These data are consistent with the findings of the study of IBs solubility in SDS. At the same time, urea is apparently a milder denaturing agent regarding the recombinant protein than SDS, as in 4 M urea, compared to 0.05% SDS (the minimum concentrations of denaturants providing, according to the data turbidimetry, complete dissolution of IBs), the recombinant protein retains its activity for a longer time. The retention of fluorescent activity by the recombinant protein solubilized from IBs with a short exposure in 0.05% SDS and 4 M urea suggests that the intermolecular interactions stabilizing IBs are less resistant to the denaturant than intramolecular contacts that provide the native conformation and activity of GFP. These results suggest that it is possible to select such solubilization conditions when the network of intermolecular contacts in IBs will be specifically disrupted, without denaturation of the correctly folded protein incorporated into the aggregates. As was demonstrated, maintaining a native-like secondary structure of the protein during its solubilization from IBs increases the yield of the recombinant protein during its refolding [27].

#### *2.4. The Proteinase K Digestion of the IBs*

PldA-GFP IBs were treated with proteinase K. Kinetics of proteolytic digestion of IBs was monitored by the measurement of turbidity at 350 nm and GFP fluorescence emission of the suspension over time. As it is shown in Figure 7, the turbidity of the IBs suspension decreases to its lowest level after 30 min of incubation, while the GFP fluorescence of the fusion protein remains practically unchanged.

**Figure 7.** Proteinase K digestion of PldA-GFP IBs. Profiles of IBs fluorescence (circle) and turbidity (square).

The results presented here strongly suggest that more than one chimeric protein species with different protease resistance coexist within IBs. Non-fluorescent species of the recombinant protein, which are highly sensitive to proteolysis, appear to be misfolded or partially folded polypeptides. Proteinase K showed a strong preference for hydrolyzing unstructured protein regions [28].

The resistance of the active protein to proteinase K digestion suggests that it has a compact supramolecular structure, as the quaternary structure of GFP has been shown to be highly resistant to proteases [26]. However, it is also possible that proteinase K partially cleaves active protein, including GFP, without affecting the chromophore. As is known, the intensity of fluorescence of the chromophore can be changed insignificantly with major disturbances of the native structure of GFP [29]. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized polypeptides susceptible to proteolysis can coexist in PldA-GFP IBs.
