A novel class of micro-electrodes was fabricated by synthesizing high density carbon nanotube islands on lithographically defined, passivated titanium nitride conductors on a silicon dioxide substrate. Electrochemical characterization in phosphate buffered saline of these new electrodes reveals superb electrochemical properties marked by featureless rectangular cyclic voltammetry curves corresponding to a DC surface specific capacitance and a volume specific capacitance as high as 10 mF cm(-2) and 10 F cm(-3), respectively. These electrodes are also characterized by a slowly varying impedance magnitude over the range of 1 Hz to 20 kHz. High fidelity extracellular recordings from cultured neurons were performed and analysed to validate the effectiveness of the fabricated electrodes. The enhanced electrochemical properties of the electrodes, their flexible and simple micro-fabrication preparation procedure as well as their bio-compatibility and durability suggest that carbon nanotube electrodes are a promising platform for high resolution capacitive electrochemical applications.
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.
The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT_TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK_Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein‐folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches. Proteins 2009. © 2009 Wiley‐Liss, Inc.
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