Globuláris és rendezetlen fehérjék, fehérje-membránmimetikum kölcsönhatások szerkezeti és dinamikai jellemzése NMR spektroszkópiával
Sebák Fanni
Pharmaceutical Sciences
Dr. Zelkó Romána
SE Egyetemi Gyógyszertár Gyógyszerügyi Szervezési Intézet
2022-05-11 11:30:00
Modern Trends in Pharmaceutical Scientific Research
Dr. Antal István
Dr. Bodor Andrea
Dr. Ambrus Attila egyetemi docens
Dr. Tőke Orsolya tudományos főmunkatárs
Dr. Noszál Béla professor emeritus
Dr. Mirzahosseini Arash egyetemi adjunktus
Dr. Béni Szabolcs egyetemi docens
Dr. Kövér Katalin egyetemi tanár
During my PhD l work, I performed the structural and dynamic analysis of different biosystems (disordered proteins, peptides, globular proteins, membrane mimetics) using NMR spectroscopy.
Disordered proteins contain large amounts of proline amino acids, which can occur in solution as both trans and cis isomers. In the p53 TAD model system, minor conformers were detected, and the proline isomers identified by the newly developed high-sensitivity 1Hα-detected methods. During phosphorylation by CK2 kinase, a shift in the cis-trans equilibrium in the TAD domain of p53 was observed for P47. The amount of cis-proline present in disordered proteins depends on the type of amino acids in the proline environment. Statistical analysis allows the regulation of cis-trans equilibrium by incorporating point mutations.
For peptides derived from ERD14 dehydrin and S100A4 protein, structural tendencies and the effect of fluorescent dye were examined by NMR spectroscopy. The peptides are disordered, and the carboxyfluorescein labelling does not significantly change the structure but is spatially close to the phenylalanine side chain of the peptide, which affects its ability to penetrate the cell. For phenylalanine-containing ERD-A, ERD-B, and S100, there is a significant difference in the intracellular fluorescence intensity of 5- and 6-carboxyfluorescein-containing peptides, suggesting different ability to enter. Based on the UC50 values of the peptides, the cell penetration ability of the 6-Cf peptides is better and there is a difference between the peptides, the order is as follows: ERD-B > ERD-A > ERD-C > S100. In addition to cell entry, intracellular localization also differs. The mechanism of entry based on experiments with inhibitors is endocytosis and macropinocytosis.
In the case of the SGPI-2 and SPINK1 skeletal serine protease inhibitors examined in my dissertation, the skeletal independence assumed by the Laskowski model is not satisfied. A different loop sequence between the two backbones is ideal, and loop replacement significantly reduces the affinity for chymotrypsin. In the case of less stable SGPI-2, loop replacement alters the structure of the inhibitor backbone. The 3rd β-strand becomes disordered, which is disadvantageous for the formation of enzyme binding. The stable SPINK1 backbone shows no structural change in the case of loop replacement, whereas the loop region is distorted, causing a significant loss of affinity.
