55 research outputs found
Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation
Fluorescence Decay Heterogeneity Model Based on Electron Transfer Processes in an Enzyme-Ligand Complex
The models are described for complex fluorescence decay of tyrosine in proteins involving continuous distribution of fluorescence lifetimes and electron transfer processes. We introduce the analytical decay function with a power-like term, which provides good fits to highly complex fluorescence decays. Moreover, the power-like term in the proposed decay functions is a manifestation of so-called Tsallis nonextensive statistics and is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence. The proposed decay functions were applied to analysis of fluorescence decays of tyrosine in a protein, i.e. the enzyme purine nucleoside phosphorylase from E. coli, free in aqueous solution and in the complex with formycin A (an inhibitor) and orthophosphate (a co-substrate), and demonstrated that both models reflect the enzyme-ligand interactions. Direct measure of heterogeneity of the enzyme systems is provided by a variance of fluorescence lifetime distribution. The possible number of deactivation channels and excited state mean lifetime can be easily derived without a priori knowledge of the complexity of studied system
Nucleoside triphosphate donors for nucleoside kinases:donor properties of UTP with human deoxycytidine kinase
Crystal structures of complexes of mouse thymidylate synthase crystallized with N 4 OH dCMP alone or in the presence of N 5,N 10 methylenetetrahydrofolate
Abstract
To solve the inhibition mechanism of thymidylate synthase (TS) by N4-hydroxy-dCMP (N4-OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N4-OH-dCMP [crystal A, belonging to the space group C 1 2 1, with two monomers in asymmetric unit (ASU), measured to 1.75 Ã… resolution] or both N4-OH-dCMP and N5,10
-methylenetetrahydrofolate (mTHF) (crystals B and C, both belonging to the space group C 2 2 21, each with a single monomer in ASU, measured to resolution of 1.35 Ã… and 1.17 Ã…, respectively). Whereas crystal A-based structure revealed the mTS-N4-OH-dCMP binary complex, as expected, crystals B- and C-based structures showed the enzyme to be involved in a ternary complex with N4-OH-dCMP and noncovalently bound dihydrofolate (DHF), instead of expected mTHF, suggesting the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of the one-carbon group to a hitherto unknown site and oxidation of THF to DHF. Moreover, both C(5) and C(6) inhibitor atoms showed sp3 hybridization, suggesting C(5) reduction, with no apparent indication of C(5) proton release. In accordance with our previous results, in all subunits of these structures the inhibitor molecule was identified as the anti rotamer of imino tautomer, forming, similar to deoxyuridine monophosphate, two hydrogen bonds with a conservative asparagine (mouse Asn220) side chain.</jats:p
Low-density plasma formation in aqueous biological media using sub-nanosecond laser pulses
We demonstrate the formation of low- and high-density plasmas in aqueous media using sub-nanosecond laser pulses delivered at low numerical aperture (NA = 0.25). We observe two distinct regimes of plasma formation in deionized water, phosphate buffered saline, Minimum Essential Medium (MEM), and MEM supplemented with phenol red. Optical breakdown is first initiated in a low-energy regime and characterized by bubble formation without plasma luminescence with threshold pulse energies in the range of E(p) ≈ 4–5 μJ, depending on media formulation. The onset of this regime occurs over a very narrow interval of pulse energies and produces small bubbles (R(max) = 2–20 μm) due to a tiny conversion (η < 0.01%) of laser energy to bubble energy E(B). The lack of visible plasma luminescence, sharp energy onset, and low bubble energy conversion are all hallmarks of low-density plasma (LDP) formation. At higher pulse energies (E(p) = 11–20 μJ), the process transitions to a second regime characterized by plasma luminescence and large bubble formation. Bubbles formed in this regime are 1–2 orders of magnitude larger in size [Formula: see text] due to a roughly two-order-of-magnitude increase in bubble energy conversion (η ≳ 3%). These characteristics are consistent with high-density plasma formation produced by avalanche ionization and thermal runaway. Additionally, we show that supplementation of MEM with fetal bovine serum (FBS) limits optical breakdown to this high-energy regime. The ability to produce LDPs using sub-nanosecond pulses focused at low NA in a variety of cell culture media formulations without FBS can provide for cellular manipulation at high throughput with precision approaching that of femtosecond pulses delivered at high NA
Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosophila melanogaster, deoxyribonucleoside kinases
Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human and the Drosophila melanogaster, deoxyribonucleoside kinases
The Effects of High Salt Concentrations on the Regulation of the Substrate Specificity of Human Recombinant Deoxycytidine Kinase
Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinases
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