Konformációs viszonyok és elektronszerkezet-változások vizsgálata fehérjékben = Investigation of conformational fluctuations and electronic structure variations in proteins

Feltérképeztük a foszfoglicerát kináz molekuláris felismerési folyamataiban szerepet játszó konformációs változásokat. Elvégeztük a foszfoinozitol kináz 3 alfa izoformájánnak 3D szerkezetpredikcióját, azonban a ligandumkötődés vizsgálatára a kooperáló partner elállása miatt nem került sor. Felderítettük a tetrahidribiopterin (BH4) kofaktor szerepe a nitrogén monoxid szintetáz (NOS) aktiválódásában. Meghatároztuk a NO kötőhelyét a nitroforin 4 (NP4) fehérje aktív helyén. Low-mode (LMOD) keresésen alapuló platfromfüggetlen konformerkereső és dokkoló eljárást dolgoztunk ki, amely az AMBER programcsomagban is hozzáférhető. Elvégeztük a kifejlesztett eljárás több konformerkereső módszerrel történő összehasonlító vizsgálatát. Eljárást dolgoztunk ki humán P450 2C9 ligandumok azonosítására és az izoforma specificitás vizsgálatára a 2C családban. Felderítettük a NADH kofaktor és NO kölcsönhatás szerepét a P450 NO-reduktáz fehérjében. A támogatott időszak alatt megkezdett hisztamin receptorok kutatását érintő vizsgálataink alapján új lipofil zsebet találtunk a H1 receptorban, ligandum információkkal segített homológiamodellezéssel előállítottuk a H4 receptor első atomi felbontású modelljét és eljárást dolgoztunk ki új H4 ligandunok azonosítására. Utóbbi eljárást az eddig ismert legkiterjedtebb szerkezet alapú szűrővizsgálatban alkalmazva új, kísérletileg is megerősített H4 ligandumokat azonosítottunk. | Conformational motions responsible for the substrate recognition in phosphoglycerate kinase have been explored. Homology model for phosphoinositol 3 kinase alpha isoform has been developed, however docking studies were suspended due to the changed interest of the partner (ComGenex Inc). The role of tetrahydrobiopterin cofactor in nitric oxide synthase has been clarified. The putative nitric oxide binding site in nitrophorin 4 has been identified. A platform-independent LMOD conformational search method has been developed and integrated to AMBER package at UCSF. The performance of this method has been evaluated and compared to other algorithms. A new virtual screening protocol has been developed for the identification of CYP 2C9 ligands. The protocol was useful for isofom specificity studies in the 2C family. The role of NADH-nitric oxide interaction in P450-No reductase has been investigated. A new lipophilic binding pocket in human histamine H1 receptor has been identified. The first atom-level model of human histamine H4 receptor has been constructed by ligand supported homology modelling. that was used to develop an effective virtual screening protocol. The protocol allowed us to perform the largest structure based virtual screening experiment using a screening database of more than 8 million compounds. Identified new chemical scaffolds showed submicromolar binding affinity towards the human histamine H4 receptor as revealed by experimental studies

Cellular response to efficient dUTPase RNAi silencing in stable HeLa cell lines perturbs expression levels of genes involved in thymidylate metabolism.

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2'-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated

Ellipsoidal primary of the RS CVn binary zeta And: Investigation using high-resolution spectroscopy and optical interferometry

We have obtained high-resolution spectroscopy, optical interferometry, and long-term broad band photometry of the ellipsoidal primary of the RS CVn-type binary system zeta And. Based on the optical interferometry the apparent limb darkened diameter of zeta And is 2.55 +/- 0.09 mas using a uniform disk fit. The Hipparcos distance and the limb-darkened diameter obtained with a uniform disk fit give stellar radius of 15.9 +/- 0.8 Rsolar, and combined with bolometric luminosity, it implies an effective temperature of 4665 +/- 140 K. The temperature maps obtained from high resolution spectra using Doppler imaging show a strong belt of equatorial spots and hints of a cool polar cap. The equatorial spots show a concentration around the phase 0.75. This spot configuration is reminiscent of the one seen in the earlier published temperature maps of zeta And. Investigation of the Halpha line reveals both prominences and cool clouds in the chromosphere. Long-term photometry spanning 12 years shows hints of a spot activity cycle, which is also implied by the Doppler images, but the cycle length cannot be reliably determined from the current data.Comment: 9 pages, 9 figures, accepted for A&

Anti-solar differential rotation

The differential rotation of anti-solar type detected by observations for several stars may result from a fast meridional flow. The sufficiently intensive meridional circulation may be caused by large-scale thermal inhomogeneities or, perhaps, by tidal forcing from a companion star. First results of simulations of the anti-solar rotation of a giant star with magnetically induced thermal inhomogeneities are presented. Perspectives for observational check of the theory are discussed.Comment: 5 pages, 6 figures, Astron. Nachr. 325, 496 (2004

Differential rotation on the lower main sequence

We compute the differential rotation of main sequence stars of the spectral types F, G, K, and M by solving the equation of motion and the equation of convective heat transport in a mean-field formulation. For each spectral type the rotation rate is varied to study the dependence of the surface shear on this parameter. The resulting rotation patterns are all solar-type. The horizontal shear turns out to depend strongly on the effective temperature and only weakly on the rotation rate. The meridional flow depends more strongly on the rotation rate and has different directions in the cases of very slow and very fast rotation, respectively.Comment: 4 pages, 5 figures, Astron. Nachr. 326, 265 (2005

Polar branches of stellar activity waves: dynamo models and observations

[Abridged abstract:] Stellar activity data provide evidence of activity wave branches propagating polewards rather than equatorwards (the solar case). Stellar dynamo theory allows polewards propagating dynamo waves for certain governing parameters. We try to unite observations and theory, restricting our investigation to the simplest mean-field dynamo models. We suggest a crude preliminary systematization of the reported cases of polar activity branches. Then we present results of dynamo model simulations which contain magnetic structures with polar dynamo waves, and identify the models which look most promising for explaining the latitudinal distribution of spots in dwarf stars. Those models require specific features of stellar rotation laws, and so observations of polar activity branches may constrain internal stellar rotation. Specifically, we find it unlikely that a pronounced poleward branch can be associated with a solar-like internal rotation profile, while it can be more readily reproduced in the case of a cylindrical rotation law appropriate for fast rotators. We stress the case of the subgiant component of the active close binary HR 1099 which, being best investigated, presents the most severe problems for a dynamo interpretation. Our best model requires dynamo action in two layers separated in radius. Observations of polar activity branches provide valuable information for understanding stellar activity mechanisms and internal rotation, and thus deserve intensive observational and theoretical investigation. Current stellar dynamo theory seems sufficiently robust to accommodate the phenomenology.Comment: 13 pages, 10 figures, 4 tables, accepted by Astronomy and Astrophysic

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

The effect of magnetic activity saturation in chromospheric flux-flux relationships

We present a homogeneous study of chromospheric and coronal flux-flux relationships using a sample of 298 late-type dwarf active stars with spectral types F to M. The chromospheric lines were observed simultaneously in each star to avoid spread due to long term variability. Unlike other works, we subtract the basal chromospheric contribution in all the spectral lines studied. For the first time, we quantify the departure of dMe stars from the general relations. We show that dK and dKe stars also deviate from the general trend. Studying the flux-colour diagrams we demonstrate that the stars deviating from the general relations are those with saturated X-ray emission and that those stars also present saturation in the H$\alpha$ line. Using several age spectral indicators, we show that they are younger stars than those following the general relationships. The non-universality of flux-flux relationships found in this work should be taken into account when converting between fluxes in different chromospheric activity indicators.Comment: Accepted for publication in the Monthly Notices of the Royal Astronomical Societ