| Abstract [eng] |
The purpose of this thesis is to develop methods that allow to determine the volumes of protein-ligand interactions, which is an important but rarely researched thermodynamic characteristic. Determination of binding volume could be used in the development of anticancer drugs, targets of which could be proteins used in this research for example Human Chaperon Protein 90 (HSP90) or Carbonic anhydrases. In this thesis protein-ligand interaction was researched using high pressure fluorimetry, changes in the fluorescence spectra of carbonic anhydrases that appear during conformation change in high pressure were analyzed. Melting pressures of I, II and XIII carbonic anhydrase isoforms and their relation to denaturant concentration were determined. By changing the concentrations of ligand and denaturant the binding volume of acetazoleamide to carbonic anhydrase II was determined. Also nuclear magnetic resonanse was used to research HSP90$\alpha$N-terminal domain conformation changes and ligand binding in high pressure. Using this method amino acids, that are first to change their environment during pressure denaturation, were identified. Also amino acids that are important in ligand binding were identified. By conducting experiments in high pressure using varying ligand concentrations the binding volume of an inhibitor ICPD9 was calculated. |
