Thomas O'Brien

Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases

A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.

Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogenous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

Escherichia Coli Pyruvate Oxidase: Interaction of a Peripheral Membrane Protein with Lipids

Pyruvate oxidase is one of several membrane-associated flavoprotein dehydrogenases that feed electrons into the Escherichia coli aerobic respiratory chain. Unlike the other flavoprotein dehydrogenases from E. coli , it is a peripheral membrane protein and can be released from the membrane by sonication. The purified enzyme is a tetramer of identical subunits. Each subunit contains a tightly bound flavin adenine dinucleotide (FAD) coenzyme. The addition of a second cofactor, thiamin pyrophosphate (TPP), is required to elicit catalytic activity. The reaction catalyzed is the oxidative decarboxylation of pyruvate to acetate and carbon dioxide. The purified enzyme is free of lipids. The enzymatic activity of pyruvate oxidase is stimulated 25-fold by the addition of any of a wide variety of amphiphiles, including phospholipids, to the assay mixture. The affinity between a charged detergent and pyruvate oxidase increases with the length of its hydrocarbon chain, suggesting that the mode of interaction between the protein and the amphiphile is primarily hydrophobic (1). In addition to the dramatic effect on the turnover number, the presence of lipid alters the Km and Hill coefficients for substrate and cofactor (2). Equilibrium binding studies with TPP have shown that the influence of lipids and detergents on the Km and Hill coefficients of this cofactor directly reflects changes in its equilibrium binding isotherm (3). Thus, the activity of this enzyme can be modulated by lipids through alteration of the strength and cooperativity of substrate and cofactor binding as well as by changing the turnover number. Not only do lipids have a dramatic influence on the catalytic events involving substrate and cofactor, but the catalytic ligands also have a strong influence on the affinity of the protein for lipids, as required by thermodynamic reciprocity. Several experiments have indicated an energetic and conformational coupling between the catalytic active site and the lipid binding site on pyruvate oxidase (2, 4, 5). The bound FAD is reduced in the presence of pyruvate and TPP. This reduction of the flavoprotein is accompanied by a conformational change that alters the solution behavior of the protein and exposes a region of the protein to proteolytic attack. The reduced

Facilitating Single-Transverse-Mode Lasing in VCSELs via Patterned Dielectric Anti-Phase Filters

A novel method to achieve single-fundamental-mode lasing and higher order mode suppression using a multi-layer, patterned, dielectric anti-phase filter is employed on the top of oxide-confined vertical-cavity surface-emitting lasers (VCSELs). Dielectric layers are deposited and patterned on individual VCSELs in a wafer-scale process to modify (increase/decrease) the mirror reflectivity across the oxide aperture via anti-phase reflections, creating spatially dependent threshold material gain and VCSEL lasing-mode control. A 1-D plane-wave propagation method is used to calculate the dielectric layer thicknesses in each spatial region needed to facilitate or suppress lasing. A single-fundamental-mode, continuous-wave output power of 3.5 mW is achieved at a lasing wavelength of 850 nm. This additive, non-destructive method allows for mode selection at any lasing wavelength and for any VCSEL layer structure without the need for destructive etching techniques or epitaxial regrowth. It also offers the capability of a tailored filter design based on available materials and deposition methods.

4CeeD: Real-Time Data Acquisition and Analysis Framework for Material-related Cyber-Physical Environments

In this paper, we present a data acquisition and analysis framework for materials-to-devices processes, named 4CeeD, that focuses on the immense potential of capturing, accurately curating, correlating, and coordinating materials-to-devices digital data in a real-time and trusted manner before fully archiving and publishing them for wide access and sharing. In particular, 4CeeD consists of novel services: a curation service for collecting data from microscopes and fabrication instruments, curating, and wrapping of data with extensive metadata in real-time and in a trusted manner, and a cloud-based coordination service for storing data, extracting meta-data, analyzing and finding correlations among the data. Our evaluation results show that our novel cloud framework can help researchers significantly save time and cost spent on experiments, and is efficient in dealing with high-volume and fast-changing workload of heterogeneous types of experimental data.

Transverse mode control in proton-implanted and oxide-confined VCSELs via patterned dielectric anti-phase filters

A novel method for controlling the transverse lasing modes in both proton implanted and oxide-confined vertical- cavity surface-emitting lasers (VCSELs) with a multi-layer, patterned, dielectric anti-phase (DAP) filter is pre- sented. Using a simple photolithographic liftoff process, dielectric layers are deposited and patterned on individual VCSELs to modify (increase or decrease) the mirror reflectivity across the emission aperture via anti-phase reflections, creating spatially-dependent threshold material gain. The shape of the dielectric pattern can be tailored to overlap with specific transverse VCSEL modes or subsets of transverse modes to either facilitate or inhibit lasing by decreasing or increasing, respectively, the threshold modal gain. A silicon dioxide (SiO2) and titanium dioxide (TiO2) anti-phase filter is used to achieve a single-fundamental-mode, continuous-wave output power greater than 4.0 mW in an oxide-confined VCSEL at a lasing wavelength of 850 nm. A filter consisting of SiO2 and TiO2 is used to facilitate injection-current-insensitive fundamental mode and lower order mode lasing in proton implanted VCSELs at a lasing wavelength of 850 nm. Higher refractive index dielectric materials such as amorphous silicon (a-Si) can be used to increase the effectiveness of the anti-phase filter on proton implanted devices by reducing the threshold modal gain of any spatially overlapping modes. This additive, non-destructive method allows for mode selection at any lasing wavelength and for any VCSEL layer structure without the need for semiconductor etching or epitaxial regrowth. It also offers the capability of designing a filter based upon available optical coating materials.

Mode Behavior of VCSELs With Impurity-Induced Disordering

This letter explores the modal behavior of oxide-confined vertical-cavity surface-emitting lasers (VCSELs) with varying emission apertures defined by impurity-induced disordering (IID) via closed ampoule zinc diffusion. A 1-D plane wave propagation method is used to calculate the mirror loss as a function of IID strength and depth. The devices are fabricated with masked areas ranging from approximately 70%–110% of the oxide aperture defining an unmodified emission aperture designed to overlap mainly with the fundamental mode. An analysis of the transverse mode lasing characteristics and mode-dependent thermal characteristics demonstrates a decrease in thermal performance associated with the increasing overlap between the IID ring and supported optical modes of the VCSEL cavity. A single-mode output power of 1.6 mW with over 30 dBm side-mode suppression ration is achieved from a <inline-formula> <tex-math notation=LaTeX>

Regulation by lipids of cofactor binding to a peripheral membrane enzyme: binding of thiamin pyrophosphate to pyruvate oxidase.

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Role of Arginine in the Binding of Thiamin Pyrophosphate to Escherichia coli Pyruvate Oxidase

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Mode Control in Vertical-Cavity Surface-Emitting Lasers

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