7 research outputs found
Interaction of ruthenium complexes with biomolecules
Jednym z najnowszych kierunków badań mających na celu poszukiwanie innowacyjnych leków antynowotworowych jest zastosowanie w chemioterapii kompleksów rutenu. Celem zrealizowanej pracy było zbadanie oddziaływania dwóch kompleksów rutenu [Ru(dip)2bpy]Cl2 oraz Ru[(DMSO)2(glu)TSC)Cl2] z trzema białkami modelowymi: lizozymem, rybonukleazą A oraz onkonazą. W ramach pracy wykonano syntezę kompleksu rutenu (II) [Ru(dip)2bpy]Cl2, przeprowadzono pomiary spektroskopowe tego związku z lizozymem i RNazą A oraz wykonano miareczkowanie fluorescencyjne badanych białek z syntezowanym kompleksem w celu zbadania ewentualnego zajścia efektu „light switch”. W części strukturalnej wykonano optymalizację krystalizacji lizozymu, RNazy A i onkonazy, a następnie przeprowadzono nasączanie i kokrystalizację z kompleksami rutenu. Przeprowadzono pomiary dyfraktometryczne, a następnie rozwiązano i udokładniono struktury lizozymu i RNAzy A krystalizowanych w obecności kompleksów rutenu. Badania spektroskopowe wykazały brak efektu „light switch” podczas oddziaływań z lizozymem i RNazą A. Otrzymane dane dyfraktometryczne ujawniły, że w kryształach białek w obecności kompleksów nie dochodzi do specyficznego wiązania związków rutenu lub ich fragmentów do białek, a jedynie do niespecyficznych oddziaływań prawdopodobnie na powierzchni ich cząsteczek.One of the current topics in innovative anticancer drugs development concerns ruthenium complexes that could be used in chemotherapy.The aim of presented work was to study the interaction between two ruthenium compounds [Ru(dip)2bpy]Cl2 and Ru[(DMSO)2(glu)(TSC)Cl2] and three model proteins: lysozyme, ribonuclease A and onconase. Ruthenium complex [Ru(dip)2bpy]Cl2 has been synthesized and studied with lysozyme and ribonuclease A using absorption and emission spectroscopy. In the context of structural studies were performed. Crystallization optimization for proteins and proteins in presence of two ruthenium compounds. To obtain crystals of protein-ruthenium complexes soaking and cocrystallization procedures were used. Obtained crystals have been investigated by X-ray diffraction and crystal structures have been solved and refined.Spectroscopic studies showed no "light switch" effect during interactions with lysozyme and RNase A which has been manifested by a decrease in fluorescence upon addition of proteins to ruthenium complexes solutions. X-ray diffraction studies revealed that there is no specific binding between ruthenium complexes or its smaller fragments and studied proteins. Probably ruthenium complexes bound in a nonspecific way to protein surface
Aqueous behavior and reactivity towards nitric oxide of NAMI-A type complexes bearing bulky N-heterocyclic ligands
The efficient -dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans
Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization
Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new putative membrane-associated domain and proton-relay system in catalysis
3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid 1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium substituted substrates. The role of the active site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modelling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modelling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation
Structure, mutagenesis, and QM:MM modeling of 3-ketosteroid -dehydrogenase from Sterolibacterium denitrificans : the role of a new putative membrane-associated domain and proton-relay system in catalysis
3-Ketosteroid Δ1-dehydrogenases (KstD)
are important
microbial flavin enzymes that initiate the metabolism of steroid ring
A and find application in the synthesis of steroid drugs. We present
a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated
domain and extended proton-relay system. The experimental and theoretical
studies show that the steroid Δ1-dehydrogenation
proceeds according to the Ping–Pong bi–bi kinetics and
a two-step base-assisted elimination (E2cB) mechanism. The mechanism
is validated by evaluating the experimental and theoretical kinetic
isotope effect for deuterium-substituted substrates. The role of the
active-site residues is quantitatively assessed by point mutations,
experimental activity assays, and QM/MM MD modeling of the reductive
half-reaction (RHR). The pre-steady-state kinetics also reveals that
the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction
(OHR), while the RHR exhibits a slight optimum at the pH usual for
the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity
of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be
the best substrate of AcmB in terms of ΔG of
binding and predicted rate of dehydrogenation