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Thermostability of lysozyme amyloid fibrils

Ordered protein aggregates, amyloid fibrils, are a marker of many serious diseases, such as Alzheimer’s, Parkinson’s, prion diseases, etc. At present, special attention is paid to the study of external influences that can affect the structure and stability of mature amyloid fibrils, which may be in demand in the development of approaches to the therapy of amyloidosis, as well as in the creation of new high-strength materials on the basis of these protein aggregates. An external factor, the influence of which on fibrils was studied in this work, was temperature denaturation. It was shown that heating lysozyme amyloid fibrils to 60 °C does not lead to their degradation, but leads only to a reversible increase in the intramolecular mobility of amyloid-forming proteins, but does not change their morphology. At the same time, boiling of lysozyme amyloids leads to their irreversible degradation, which occurs at least 5 days after exposure: fibrils that form larger clusters change their secondary structure, and fibrils with a lesser degree of clustering are divided into separate fibers. Obtained data about the factors that change the stability and structure of amyloids can be applied in biotechnology for creating new high-strength nanomaterials on their basis.


Introduction
Highly ordered protein aggregates, amyloid fibrils, are a marker of many serious diseases, such as Alzheimer's, Parkinson's, prion diseases, and others [1,2]. At the same time, physiological amyloid fibrils in bacteria, unicellular eukaryotes, fungi, plants, insects and mammals were found, which are necessary for the normal functioning of cells and tissues, which emphasizes the biological significance of amyloid formation [3,4]. Due to the widespread occurrence of amyloid fibrils, as well as the variety of their functions (mechanical protection and modification of the cell surface; biotic or abiotic surface adhesion; cellular internalization; ensuring resistance to various external influences; biosynthesis of pigments, control of homeostasis, storage and the release of hormones, signal transduction, etc. [5,6]) the study of amyloids is a very urgent task. The relevance of amyloid fibrils study is also due to their unique mechanical properties, such as high strength and extensibility, which makes them an attractive object for the creation of new nanostructures and nanomaterials [7]. The aim of this work was to analyze the resistance of amyloid fibrils to high temperatures. The object of the study was model amyloid fibrils formed from the globular protein lysozyme with different morphology.

Experimental techniques
Fibrillogenesis of lysozyme was carried out using two different protocols, making it possible to obtain aggregates with different morphologies [8]. According to one of these protocols, lysozyme was 2 incubated in 20% acetic acid in the presence of 100 mM NaCl (pH 2) at 37 °C. According to the second protocol, the protein was incubated in 0.05 M KH2PO4-NaOH buffer in the presence of 3 M GdnHCl (pH 7) at 57 °C. Both samples were incubated with constant stirring for 2 days. Then the fibrils were transferred to distilled water. The prepared amyloid fibrils were studied by the absorption and fluorescence spectroscopy, method of circular dichroism and electron microscopy. In addition, using a specially developed approach based on the samples preparation by equilibrium microdialysis [9, 10, and 11], the interaction with amyloids of a specific fluorescent probe thioflavin T (ThT) was studied. The recorded fluorescence intensity of the samples was corrected on the primary inner filter effect according to a specially elaborated protocol [12].

Results
According to electron microscopy data, amyloid fibrils prepared at pH 2 predominantly are thin fibers, having a tendency to form clots. At the same time, in the buffer solution with pH 7 shorter fibrils with a significantly higher degree of clustering are formed ( Figure 1). First of all, we investigated the stability of the prepared amyloid fibrils when changing temperature in the range from 0 to 60 ºС. The fluorescence spectra of the sample (free and bound to fibrils ThT) and reference (free ThT) solutions obtained by the equilibrium microdialysis were recorded when they were heated from 0 to 60 °C. It turned out that heating the prepared samples leads to a decrease in the fluorescence intensity of ThT bound to fibrils. In order to check whether this decrease is reversible, we cooled the samples to 0 °C. The curve characterizing the heating of the samples almost completely coincided with the curve characterizing their cooling for both samples (Figure 2), which made it possible to conclude that the processes occurring in the samples when they are heated to 60 °C are reversible.
In order to understand what exactly is responsible for the decrease in the ThT fluorescence intensity with heating the sample, we analyzed images of amyloid fibrils obtained by electron microscopy before and after external exposure. We found no changes in the structure or degradation of amyloids after their heating to 60 °C (Figures 3, 4).
It can be assumed that an increase in the sample temperature increases the probability of the ThT molecule transition to the conformation with an angle between the benzothiazole and aminobenzene rings equal to 90 °C, which, in turn, leads to nonradiative deactivation of the excited state of the dye and a decrease in the quantum yield of its fluorescence. This can occur due to a reversible change in the intramolecular mobility of side radicals of amyloid-forming proteins and fragments of the ThT molecule relative to each other, without changing the structure or degradation of amyloid fibrils. Thus, obtained results indicate that lysozyme amyloid fibrils prepared under different conditions are stable     Electron microscopy data of the samples a week after boiling indicate that boiling lysozyme amyloid fibrils, prepared at pH 2, leads to their partial degradation: declusterization of not numerous fibrils clots into the separate thin filaments ( Figure 6) and to a slight change in their secondary structure, which was evaluated by the recorded circular dichroism (CD) spectra of the samples in the far UV region (Figure 7 a).   The boiling of lysozyme amyloid fibrils prepared at pH 7 also led to their degradation, which, however, was more manifested in a change in the secondary structure of amyloid-forming proteins (Figure 7 b), but practically did not lead to declusterization of fibrillar clots (Figure 8).

Conclusion
Based on the obtained results, it was concluded that heating lysozyme amyloid fibrils with different morphology up to 60 °С causes only a reversible increase in the intramolecular mobility of amyloidforming proteins. At the same time, boiling of lysozyme amyloids leads to their irreversible degradation: amyloids that form larger clusters change their secondary structure, while fibrils with a lesser degree of clustering are divided into separate fibers. New data about the factors that influence the stability of amyloids can be applied in biotechnology for creating new high-strength nanomaterials on their basis.