La minorite bulgare de Roumanie: entre conservation de l'identite ethnique et emergence de la representation politique apres 1989

The present article summarises the results of empirical research conducted in the two Bulgarians' communities in Romania. The general problems are declined in two axes, historical and political. The historical approach aims at showing the coming of Bulgarians to Romania and briefly maps and follows the various waves of immigration. For its part, the political approach analyzes the current political situation. The article is a valuable empirically-based contribution to the study of the processes of integration of the national minorities in Romania

Conformational effects on the Circular Dichroism of Human Carbonic Anhydrase II: a multilevel computational study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions

Conformational flexibility influences structure–function relationships in tyrosyl protein sulfotransferase-2

Tyrosine sulfation is a very important posttranslational modification of proteins. It is catalyzed by tyrosylprotein sulfotransferase and recently became increasingly important for biomedicine and pharmacy. An important insight about structure–activity relationships of human tyrosylprotein sulfotransferase has been received by elucidating the crystal structure, but there is still no understanding about how conformational flexibility and dynamics which are fundamental protein properties influence structure–function relationships of the enzyme. In order to provide this missing but crucially important knowledge we performed a comprehensive atomistic molecular dynamics study which revealed that (i) the conformational flexibility influences sensitively key structural determinants and interactions between the enzyme, the substrate and the cofactor; (ii) a more open conformation adopted by the substrate for binding in TPST 2; (iii) the mutations of key residues related with catalysis and binding change alter the enzyme structure and influence important interactions between the enzyme, the cofactor and the substrate

Conformational effects on the pro-S hydrogen abstraction reaction in cyclooxygenase-1: an integrated QM/MM and MD study

A key step in the cyclooxygenase reaction cycle of cyclooxygenase 1 (COX-1) is abstraction of the pro-S hydrogen atom of the arachidonic acid by a radical that is formed at the protein residue Tyr-385. Here we investigate this reaction step by a quantum-mechanics/molecular-mechanics approach in combination with molecular-dynamics simulations. The simulations identify the hydrogen abstraction angle as a crucial geometric determinant of the reaction, thus revealing the importance of the cyclooxygenase active site for calculating the potential energy surface of the reaction

Unusual catalytic strategy by non-heme Fe(ii)/2-oxoglutarate-dependent aspartyl hydroxylase AspH

Biocatalytic C-H oxidation reactions are of important synthetic utility, provide a sustainable route for selective synthesis of important organic molecules, and are an integral part of fundamental cell processes. The multidomain non-heme Fe(ii)/2-oxoglutarate (2OG) dependent oxygenase AspH catalyzes stereoselective (3R)-hydroxylation of aspartyl- and asparaginyl-residues. Unusually, compared to other 2OG hydroxylases, crystallography has shown that AspH lacks the carboxylate residue of the characteristic two-His-one-Asp/Glu Fe-binding triad. Instead, AspH has a water molecule that coordinates Fe(ii) in the coordination position usually occupied by the Asp/Glu carboxylate. Molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies reveal that the iron coordinating water is stabilized by hydrogen bonding with a second coordination sphere (SCS) carboxylate residue Asp721, an arrangement that helps maintain the six coordinated Fe(ii) distorted octahedral coordination geometry and enable catalysis. AspH catalysis follows a dioxygen activation-hydrogen atom transfer (HAT)-rebound hydroxylation mechanism, unusually exhibiting higher activation energy for rebound hydroxylation than for HAT, indicating that the rebound step may be rate-limiting. The HAT step, along with substrate positioning modulated by the non-covalent interactions with SCS residues (Arg688, Arg686, Lys666, Asp721, and Gln664), are essential in determining stereoselectivity, which likely proceeds with retention of configuration. The tetratricopeptide repeat (TPR) domain of AspH influences substrate binding and manifests dynamic motions during catalysis, an observation of interest with respect to other 2OG oxygenases with TPR domains. The results provide unique insights into how non-heme Fe(ii) oxygenases can effectively catalyze stereoselective hydroxylation using only two enzyme-derived Fe-ligating residues, potentially guiding enzyme engineering for stereoselective biocatalysis, thus advancing the development of non-heme Fe(ii) based biomimetic C-H oxidation catalysts, and supporting the proposal that the 2OG oxygenase superfamily may be larger than once perceived

Structural Insights from Molecular Dynamics Simulations of Tryptophan 7-Halogenase and Tryptophan 5-Halogenase

Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenasesPrnA and PyrHwas investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site

Dimerization and ligand binding in tyrosylprotein sulfotransferase-2 are influenced by molecular motions

Tyrosylprotein sulfotransferase-2 catalyses important, but a less explored posttranslational modification of proteins. The crystallographic structure of the full complex, containing the enzyme, PAPS cofactor and the peptide substrate (WTFC) revealed important structural and atomistic details about the overall structure, binding sites and interactions in TPST-2, but is not informative about the conformational flexibility, which is a fundamental protein property. In a recent computational study we analyzed the impact of conformational flexibility on key geometric determinants and interactions in WTFC. However, there are still unexplained effects about how the conformational dynamics influences the formation of the catalytically active dimer form of the enzyme and also how the binding of the cofactor and the substrate to the apoenzyme influences the structure and the pattern of correlated motions in the enzyme. In order to provide the missing knowledge we performed a molecular dynamics study on the dimerization mutant W113A, the apoenzyme (APO), enzyme–cofactor complex (ES), and enzyme–substrate complex (ES) and compared the results to the WTFC. We identified new hydrophobic interactions important for the stabilization of the enzyme dimer and provided structural insight about the role of the key mutation W113A for the dimerization in the absence of experimentally-derived structure. In addition we showed that the binding of the substrate and cofactor to the apoenzyme contributes to the stability of the whole active complex, influences the local interactions in the binding site and importantly, affects the pattern of the correlated motions in the entire molecule

Unusual catalytic strategy by non-heme Fe(ii)/2-oxoglutarate-dependent aspartyl hydroxylase AspH

Biocatalytic C-H oxidation reactions are of important synthetic utility, provide a sustainable route for selective synthesis of important organic molecules, and are an integral part of fundamental cell processes. The multidomain non-heme Fe(ii)/2-oxoglutarate (2OG) dependent oxygenase AspH catalyzes stereoselective (3R)-hydroxylation of aspartyl- and asparaginyl-residues. Unusually, compared to other 2OG hydroxylases, crystallography has shown that AspH lacks the carboxylate residue of the characteristic two-His-one-Asp/Glu Fe-binding triad. Instead, AspH has a water molecule that coordinates Fe(ii) in the coordination position usually occupied by the Asp/Glu carboxylate. Molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies reveal that the iron coordinating water is stabilized by hydrogen bonding with a second coordination sphere (SCS) carboxylate residue Asp721, an arrangement that helps maintain the six coordinated Fe(ii) distorted octahedral coordination geometry and enable catalysis. AspH catalysis follows a dioxygen activation-hydrogen atom transfer (HAT)-rebound hydroxylation mechanism, unusually exhibiting higher activation energy for rebound hydroxylation than for HAT, indicating that the rebound step may be rate-limiting. The HAT step, along with substrate positioning modulated by the non-covalent interactions with SCS residues (Arg688, Arg686, Lys666, Asp721, and Gln664), are essential in determining stereoselectivity, which likely proceeds with retention of configuration. The tetratricopeptide repeat (TPR) domain of AspH influences substrate binding and manifests dynamic motions during catalysis, an observation of interest with respect to other 2OG oxygenases with TPR domains. The results provide unique insights into how non-heme Fe(ii) oxygenases can effectively catalyze stereoselective hydroxylation using only two enzyme-derived Fe-ligating residues, potentially guiding enzyme engineering for stereoselective biocatalysis, thus advancing the development of non-heme Fe(ii) based biomimetic C-H oxidation catalysts, and supporting the proposal that the 2OG oxygenase superfamily may be larger than once perceived

Role of structural dynamics in selectivity and mechanism of non-heme Fe(II) and 2-Oxoglutarate-dependent Oxygenases involved in DNA repair

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic β-hairpin, are involved in substrate binding in AlkBH2–dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)═O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2–dsDNA and AlkB–dsDNA; in AlkB–ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein–DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism

How does conformational flexibility influence key structural features involved in activation of anaplastic lymphoma kinase?

Anaplastic Lymphoma Kinase (ALK) plays a major role in developing tumor processes and therefore has emerged as a validated therapeutic target. Applying atomistic molecular dynamics simulations on the wild type enzyme and the nine most frequently occurring and clinically important activation mutants we revealed important conformational effects on key interactions responsible for the activation of the enzyme