3.16.3. Fluorescence Spectra

The ThT and GFP fluorescence spectra were recorded on a RF-5301 PC spectrofluorophotometer (Shimadzu, Japan) in quartz cuvettes with an optical path length of 1 cm in the range from 460 to 650 nm at λex = 450 nm. The excitation and emission slit widths were set at 5 nm. The measurements of GFP fluorescence activity of the recombinant proteins were performed in the range of low concentrations (0.1–1.1 mg/mL), where the intensity of GFP fluorescence is directly proportional to its concentration. For comparison, all the GFP fluorescence data were normalized by the maximum fluorescence signal.

#### **4. Conclusions**

Despite their fundamental and practical importance, IBs are not well understood. To date, there are many unresolved and controversial issues regarding the conformational states and arrangement of protein molecules inside the IBs, as well as the exact nature of intermolecular interactions. Various biophysical methods have been used to characterize proteins aggregated in IBs, including NMR one, which enables detailed structural information at the specific residue level up to the three-dimensional structure of the protein to be obtained. However, no significant progress has been made in these studies so far.

Here, we used GFP as a fusion tag to provide further opportunities for structural studies of IBs. Therefore, some details of the inner organization IBs were revealed. The data obtained indicate the presence of highly structured clusters of correctly folded protein molecules inside the IBs, which can differ from one another in the degree of order in the structure. Intramolecular interactions that maintain the native-like conformation of the protein in these clusters may be more resistant to denaturants than intermolecular contacts in IBs.

According to our data, PldA-GFP IBs formed at low temperatures contain a noticeable percentage of the protein in the native conformation, and the recombinant protein solubilized from IBs has a high content of β-structure. This fact suggests that IB formation occurs at a relatively late stage of protein folding, when the degree of protein structural organization is high enough and proteins retain most of their secondary structure during aggregation. The results of the structural analysis of PldA-GFP IBs contribute to understanding the mechanisms of IB formation and their structure.

The accumulation of knowledge about IBs formed by proteins of different nature under various expression conditions provides a deeper understanding of their molecular organization, opening possibilities for controlling their structure and the process of formation. Further progress in the study of IBs will require new methodological approaches, as well as powerful biophysical research methods.

**Supplementary Materials:** The following are available online: Figure S1. Recombinant plasmid for the expression of the PldA-GFP fusion protein.

**Author Contributions:** S.I.B. was in charge of overall direction and planning and performed experiments (isolation and characterization of recombinant proteins); N.Y.K. carried out experiments (spectroscopic methods); A.M.S. and E.P.B. performed molecular genetic experiments; E.V.S. performed experiments (DLS, FPLC); E.S.M., T.Y.G. and N.A.S. performed bioactivity testing supervised by D.L.A.; and T.F.S. conducted project administration, literature analysis, data analysis, and interpretation, conceived the study, and was in charge of overall direction and planning. All authors discussed the results and contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** All the animal experiments were carried out in accordance with the interstate standard "Guide for the maintenance and care of laboratory animals (GOST 33215-2014)", which takes into account the main provisions of the international document ETS N 123, Appendix A of the European Convention for the protection of Vertebrate animals used for experimental and other scientific purposes Guidelines for accommodation and care of animals (Article 5 of the Convention) and in accordance with the animal care protocols and rules, approved for pre-clinical studies in Russian Federation and permitted by PIBOC Animal Ethics Committee. All animals were housed in standard facility under controlled environmental conditions at temperature 22 ± 2 ◦C and 12-h light-dark cycle, balanced ad libitum diet for laboratory animals in accordance with the Russian GOST P 50258-92 protocol and water ad libitum. 6- to 8-weeks old female BALB/c mice weighing 20 ± 2 g for the experiments were use.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**Acknowledgments:** The study was carried out on the equipment of the Collective Facilities Center «The Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS».

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

**Sample Availability:** Samples of the compounds are available by agreement with the authors and management of the organization for conducting joint research from the authors.

#### **Abbreviations**

PldA—detergent-resistant phospholipase A1; GFP—green fluorescent protein; PldA-GFP chimeric protein; IB—inclusion body; CD—circular dichroism; DLS—dynamic light scattering; LPS—lipopolysaccharide; SDS-PAGE—sodium dodecyl-sulfate polyacrylamide gel electrophoresis; ThT—Thioflavin T; RH—hydrodynamic radius; PBS—phosphate-buffered saline, pH 7.5; and IPTG isopropyl β-D-1-thiogalactopyranoside.
