Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Br?nsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Br?nsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.     

An ESR and ENDOR study of irradiated 6Li-formate

Lithium formate ((6)LiOOCH.H(2)O), 95% (6)Li enrichment, combined with an exchange of crystallization water with D(2)O was investigated. The ESR spectrum of the radiation induced free radicals stable at room temperature consists of a singlet with a narrow line width, 0.92mT. (6)Li has smaller magnetic moment and nuclear spin, which resulted in the narrower line width accompanied with an increase in peak amplitude. In comparison with lithium formate with natural isotopic composition, (6)Li (7.5%, I=1) and (7)Li (92.5%, I=3/2), the sensitivity was increased by a factor of two. With optimised spectrometer settings (6)Li formate had seven times higher sensitivity compared to alanine. Therefore this material is proposed as a dosimeter material in a dose range down to 0.1Gy. The g and the (13)C-hyperfine (hf) tensors of the CO(2)(-) radical anion, major paramagnetic products, were evaluated to be g=(2.0037, 1.9975, 2.0017), and A((13)C)=(465.5, 447.5, 581.3) MHz for polycrystalline samples at room temperature. Furthermore, the (1)H-hf and (6)Li-hf tensors observed for the surroundings of CO(2)(-) by ENDOR technique were in fairly good agreement with DFT calculations. The CO(2)(-) radicals are found to be so stable that the formate is applicable to the ESR dosimetry, because of fully relaxing in a fully relaxed geometrical structure of the CO(2)(-) component and remaining tight binding with the surroundings after the H atom detachment from HCO(2)(-).     

Structures of the hexafluorocyclopropane, octafluorocyclobutane, and decafluorocyclopentane radical anions probed by experimental and computational studies of anisotropic electron spin resonance (ESR) spectra

Anisotropic electron spin resonance (ESR) spectra are reported for the radical anions of hexafluorocyclopropane (c-C(3)F(6)(-)), octafluorocyclobutane (c-C(4)F(8)(-)), and decafluorocyclopentane (c-C(5)F(10)(-)) generated via gamma-irradiation in plastically crystalline tetramethylsilane (TMS) and rigid 2-methyltetrahydrofuran (MTHF) matrices. By combining the analysis of these experimental ESR spectra involving anisotropic hyperfine (hf) couplings with a series of quantum chemical computations, the geometrical and electronic structure of these unusual perfluorocycloalkane radical anions have been characterized more fully than in previous studies that considered only the isotropic couplings. Unrestricted Hartree-Fock (UHF) computations with the 6-311+G(d,p) basis set predict planar ring structures for all three radical anions, the ground electronic states being (2)A(2)(") for c-C(3)F(6)(-) (D(3h) symmetry), (2)A(2u) for c-C(4)F(8)(-) (D(4h)), and (2)A(2)(") for c-C(5)F(10)(-) (D(5h)), in which the respective six, eight, and ten 19F-atoms are equivalent by symmetry. A successful test of the theoretical computation is indicated by the fact that the isotropic 19F hf couplings computed by the B3LYP method with the 6-311+G(2df,p) basis set for the optimized geometries are in almost perfect agreement with the experimental values: viz., 19.8 mT (exp) vs 19.78 mT (calc) for c-C(3)F(6)(-); 14.85 mT (exp) vs 14.84 mT (calc) for c-C(4)F(8)(-); 11.6 mT (exp) vs 11.65 mT (calc) for c-C(5)F(10)(-). Consequently, the same computation method has been applied to calculate the almost axially symmetric anisotropic 19F hf couplings for the magnetically equivalent 19F atoms: (-4.90 mT, -4.84 mT, 9.75 mT) for c-C(3)F(6), (-3.54 mT, -3.48 mT, 7.02 mT) for c-C(4)F(8)(-), and (-2.62 mT, -2.56 mT, 5.18 mT) for c-C(5)F(10)(-). ESR spectral simulations performed using the computed principal values of the hf couplings and the spatial orientations of the 19F nuclei as input parameters reveal an excellent fit to the experimental anisotropic ESR spectra of c-C(3)F(6)(-), c-C(4)F(8)(-), and c-C(5)F(10)(-), thereby providing a convincing proof of the highly symmetric D(nh) structures that are predicted for these negative ions. Furthermore, using the computed 19F principal values and their orientations, the effective 19F anisotropic hf couplings along the molecular symmetry axes were evaluated for c-C(3)F(6)(-) and c-C(4)F(8)(-) and successfully correlated with the positions of the characteristic outermost features in both the experimental and calculated anisotropic spectra. In addition, the electronic excitation energies and oscillator strengths for the c-C(3)F(6)(-) , c-C(4)F(8)(-), and c-C(5)F(10)(-) radical anions were computed for the first time using time-dependent density functional theory (TD-DFT) methods.     

Comparison of chromatographic ion-exchange resins VI. Weak anion-exchange resins

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.     

Determination of wavelength-specific UV protection factors of sunscreens in intact skin by EPR measurement of UV-induced reactive melanin radical

There remains an unmet need for skin tissue-based assays for the measurement of the UVA protection and efficacy of sunscreens. Here we describe development of a novel electron paramagnetic resonance assay that uses the photogeneration of reactive melanin radical as a measure of UV light penetration to melanocytes in situ in skin. We have used areas of focal melanocytic hyperplasia in the skin of Monodelphis domestica to model the human nevus. We show that we are able to use this assay to determine the monochromatic protection factors (mPF) of research and commercial sunscreens at specific narrow wavebands of UVB, UVA and blue visible light. Both commercial sunscreens, a sun protection factor (SPF) 4 and an SPF 30 product, had mPFs in the UVB range that correlated well with their claimed SPF. However, their mPF in the UVA ranges were only about one-third of claimed SPF. This technique can be used to design and assay sunscreens with optimally balanced UVA and UVB protection.     

Spatial distribution and decrease of dye solar cell performance induced by electrolyte filling

The spatial performance variation of dye solar cell with standard liquid electrolyte was examined by dividing the cell into segments. Surprisingly large and permanent performance differences were found in different parts of the cell leading to significant losses in the overall cell efficiency. The decrease of open circuit voltage along the electrolyte filling direction suggests that 4-tert-butylpyridine is adsorbed non-uniformly as the electrolyte passes through the dyed TiO₂ layer during the filling process. The result indicates that non-uniform electrolyte adsorption may limit the up-scaling of dye solar cells, which calls for the examination of electrolyte filling techniques and electrolyte compositions less prone to this effect.     

Optimal information acquisition for a linear quadratic control problem

This paper develops a generalization of the linear quadratic control problem with partial information. As in the standard partial information setting, it is assumed that the state variable is only observed with noise. The idea in this paper is that the information level may be chosen optimally. In real life information is costly to acquire. It is therefore a trade off between the costs of getting detailed information and the increased value this information gives. We believe that the technique we present should have potential for application within both economics and engineering.     

Concurrent multi-scale design optimization of composite frame structures using the Heaviside penalization of discrete material model

This paper deals with the concurrent multi-scale optimization design of frame structure composed of glass or carbon fiber reinforced polymer laminates. In the composite frame structure, the fiber winding angle at the micro-material scale and the geometrical parameter of components of the frame in the macro-structural scale are introduced as the inde-pendent variables on the two geometrical scales. Considering manufacturing requirements, discrete fiber winding angles are specified for the micro design variable. The improved Heaviside penalization discrete material optimization inter-polation scheme has been applied to achieve the discrete optimization design of the fiber winding angle. An optimiza-tion model based on the minimum structural compliance and the specified fiber material volume constraint has been estab-lished. The sensitivity information about the two geometrical scales design variables are also deduced considering the char-acteristics of discrete fiber winding angles. The optimization results of the fiber winding angle or the macro structural topology on the two single geometrical scales, together with the concurrent two-scale optimization, is separately studied and compared in the paper. Numerical examples in the paper show that the concurrent multi-scale optimization can fur-ther explore the coupling effect between the macro-structure and micro-material of the composite to achieve an ultra-light design of the composite frame structure. The novel two geometrical scales optimization model provides a new oppor-tunity for the design of composite structure in aerospace and other industries.     

Spectroelectrochemical cell for in situ studies of solid oxide fuel cells

Solid oxide fuel cells (SOFCs) are able to produce electricity and heat from hydrogen‐ or carbon‐containing fuels with high efficiencies and are considered important cornerstones for future sustainable energy systems. Performance, activation and degradation processes are crucial parameters to control before the technology can achieve breakthrough. They have been widely studied, predominately by electrochemical testing with subsequent micro‐structural analysis. In order to be able to develop better SOFCs, it is important to understand how the measured electrochemical performance depends on materials and structural properties, preferably at the atomic level. A characterization of these properties under operation is desired. As SOFCs operate at temperatures around 1073 K, this is a challenge. A spectroelectrochemical cell was designed that is able to study SOFCs at operating temperatures and in the presence of relevant gases. Simultaneous spectroscopic and electrochemical evaluation by using X‐ray absorption spectroscopy and electrochemical impedance spectroscopy is possible.     

The Nano-Jackhammer effect in probing near-surface mechanical properties

Because of its ease of implementation and insensitivity to indenter drift, dynamic indentation techniques have been frequently used to measure mechanical properties of bulk and thin film materials as a function of indenter displacement. However, the actual effect of the oscillating tip on the material response has not been examined. Recently, it has been shown that the oscillation used with dynamic indentation techniques alters the measured hardness value of ductile metallic materials, especially at depths less than 200 nm. The alteration in the hardness is due to the added energy associated with the oscillation which assists dislocation nucleation. Atomistic simulations on nickel thin films agree with experiments that more dislocations are nucleated during dynamic indents than with quasi-static indents. Through the analysis of quasi-static and dynamic indents made into nickel single crystals and thin films, a theory to describe this phenomenon is presented. This is coined the Nano-Jackhammer effect, a combination of dislocation nucleation and strain rate sensitivity caused by indentation with a superimposed dynamic oscillation.