The purpose of this study was to isolate small organic compounds (thermolytes) from hyperthermophilic archaeon Aeropyrum pernix K1 and research their effect on insulin aggregation/fibrillation. We started our research by isolating thermolytes with HPLC and continued to study the aggregation of insulin chains at different conditions. We isolated two small organic compounds from the archaeon, but we were unable to identify them. Aggregation of insulin chains was firstly studied using emission fluorescent spectroscopy with the dye thioflavin T (ThT). Chain B formed more fibrils at neutral than at acidic pH, while chain A fibrillated solely at pH 2.0. Measurements of circular dichroism after the fibrillation showed the formation of β-structure by chain B, which is typical for fibrils. Mannose and N-acetylglucosamine were the best inhibitors of fibrillation, glucose had small effect, glycerol only affected fibrillation at high concentration, myo-inositol did not inhibit fibrillation at all. By determining the secondary structure of polypeptides with added compounds, the formation of fibrils was confirmed or refuted. Native polyacrylamide gel electrophoresis indicated that during fibrillation some molecules of insulin chains remained in monomeric form and were not incorporated into fibrils. The molecular docking data exhibited that tested small organic compounds bind weakly to chain B. In this thesis we successfully set a method for thermolyte isolation, found out which molecules affect insulin chains fibrillation, but unfortunately, we were not able to confirm or refute the hypothesis about the effect of thermolytes on insulin aggregation.
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