Tesis doctoral de Zoran Nikolovski
Summary eosinophil cationic protein (ecp/rnase 3), which belongs to the rnase a superfamily and is involved in inflammatory processes mediated by eosinophils was studied in this thesis, especially its ribonuclease activity and structural stability. Ecp is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines. as the quantity available from natural sources was limited, for a thorough analysis it was necessary to optimize the expression system to obtain enough protein for such studies. thus, a high yield prokaryotic expression system with a synthetic gene for human ecp allowed studies of ribonuclease activity and thermostability. the catalytic efficiencies for oligouridylic acids of the type (up)nu>p, mononucleotides u>p and c>p, and dinucleoside monophosphates cpa, upa, and upg have been interpreted by the specific subsites distribution in ecp. the distribution of products derived from digestion of high molecular mass substrates, such as poly(u) and poly(c), by ecp was compared with that of rnase a. The characteristic cleavage pattern of polynucleotides by ecp suggests that an exonuclease-like mechanism is predominantly favoured in comparison to the endonuclease catalytic mechanism of rnase a. comparative molecular modelling with bovine pancreatic rnase a-substrate analogue crystal complexes revealed important differences in the subsite structure. Whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (b2) is severely impaired. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ecp and the shift from an endonuclease to an exonuclease-type mechanism. proposed mutations of ecp (w10k and loop containing b2) failed to modify catalytic efficiency, even though the predominantly exonuclease-like behaviour slightly shifted toward endonuclease behaviour. the thermal stability of ecp was studied by fourth-derivative uv absorbance spectra, circular dichroism, differential scanning calorimetry, and fourier transform infrared spectroscopy. the t1/2 values obtained with the different techniques were in very good agreement (t1/2 = 72°c), and the stability was maintained in the ph range between 5 and 7. The ecp calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a t1/2 of 44°c. Both calorimetric transitions disappeared after successive re-heatings, and the ratio deltah versus deltahvh of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ecp variants, the contribution of specific amino acids, such as trp10 and the region 115-122, to thermal stability was also determined. simulated thermal denaturation gave rise to the speculation that the region of the molecule important for the structural stability is the alfa3 helix as a region responsible for hinge-bending motions, observed in molecular dynamic studies in other rnases. Flexibility/rigidity of alfa3 helix, which is a mixed alf/310-helix and behaves as a mechanical hinge during the breathing motion of the protein, could play a crucial role in conformational motions of the enzyme molecule during catalysis.
Datos académicos de la tesis doctoral «Studies on the expression, kinetics and structure stability of eosinophil cationic protein/ribonuclease 3«
- Título de la tesis: Studies on the expression, kinetics and structure stability of eosinophil cationic protein/ribonuclease 3
- Autor: Zoran Nikolovski
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 08/09/2009
Dirección y tribunal
- Director de la tesis
- Claudi Cuchillo Foix
- Tribunal
- Presidente del tribunal: xavier Parés casasampera
- Jorge Segura (vocal)
- (vocal)
- (vocal)