Amplificador de señal analógico basado en conceptos biomiméticos. Desarrollo de modelos computacionales y validación experimental

La presente tesis doctoral estudia las características de un transductor acústico bioinspirado en la estructura del maxilar inferior de un Zifio de Couvier (Ziphius cavirostris). El mecanismo de funcionamiento del sensor se basa en las características de un sistema acoplado formado por los distintos componentes acústicos identificados en el maxilar. Para analizar las características del sensor se propone un modelo simplificado 2D que consta de una cavidad cerrada con forma de bocina acoplada a una lengüeta. Una parte de la lengüeta se encuentra dentro de la cavidad y otra en el exterior. Dicha lengüeta detecta los cambios de presión acústica y las vibraciones generadas por el sonido y las transmite con ondas de flexión al interior de la cavidad. La excitación prolongada sobre la placa puede provocar la activación de los modos propios del sistema acoplado. Dichos modos se caracterizan porque presentan un máximo de presión en el cono de la bocina la cual a su vez actúa como un amplificador acústico. Mediante el Método de los elementos Finitos se analizan las características acústicas del sensor y se construye un prototipo experimental para validar los resultados evaluados en el modelo numérico. Se propone una metodología numérica que permite desarrollar y validar un elemento tetraédrico para caracterizar el comportamiento isotrópico de los medios porosos. La metodología permite construir elementos de línea y bidimensionales. A partir de esta metodología se desarrolla un elemento plano ortotrópico. Se realiza un modelo de la cavidad en el que una de las paredes de la bocina está constituida por material poroso y se une la parte exterior de la lengüeta a dicho material para que constituya una viga sobre un apoyo elástico. Se calcula la respuesta modal y se discuten los efectos del material poroso en la eficiencia del transductor y las posibles mejoras a introducir en el mismo. SUMMARY This Thesis studies the characteristics of an acoustic transducer bioinspired by the structure of the lower maxilla of an odontoceto. In this case a Cuvier’s beaked whale (Ziphius cavirostris). The transducer working mechanism is based in a coupled system, with components identified in the maxilla. To analyze the transducer a simplified 2D model composed by a horn shaped closed cavity is modeled. The cavity is coupled with a flat belt. The belt has one part inside the cavity and the other part outside of it. The belt traverses the cavity wall and it is in charge to pick the vibrations from outside and introduce it inside the cavity. The transmission is obtained through the belt bending. A sustained external load with the right frequency contents will allow the system to reach a stationary pressure intensity distribution inside the cavity. Frequencies with modal shapes that show an important intensity increase at the horn tip are of special interest because of the signal amplification. A finite element model is constructed to study the transducer coupled modes and a prototype is constructed to validate the numerical results. A numerical methodology to construct and validate a tetrahedral finite element for isotropic porous materials is presented. The methodology allows constructing linear and 2D elements. It is extended to model orthotropic porous materials behavior. At the end, one of the horn walls is made of an orthotropic material and the external belt is glued to it in order to configure a belt over an elastic foundation. Modal response is evaluated and the porous material effects in the transducer efficiency and further improvements are discussed.

Band gap control via tuning of inversion degree in CdIn2S4 spinel

Based on theoretical arguments, we propose a possible route for controlling the band-gap in the promising photovoltaic material CdIn2S4. Our ab initio calculations show that the experimental degree of inversion in this spinel (fraction of tetrahedral sites occupied by In) corresponds approximately to the equilibrium value given by the minimum of the theoretical inversion free energy at a typical synthesis temperature. Modification of this temperature, or of the cooling rate after synthesis, is then expected to change the inversion degree, which in turn sensitively tunes the electronic band-gap of the solid, as shown here by screened hybrid functional calculations.

Electronic properties of doped magnesium thioindate ternary spinel in the normal and in the inverse structure

We present a theoretical study of the structural and electronic properties of the M-doped MgIn2S4 ternary spinel semiconductor with M = V, Cr, and Mn. All substitutions, in the normal and in the inverse structure, are analyzed. Some of these possible substitutions present intermediate-band states in the band gap with a different occupation for a spin component. It increases the possibilities of inter-band transitions and could be interesting for applications in optoelectronic devices. The contribution to, and the electronic configuration of, these intermediate bands for the octahedral and tetrahedral sites is analyzed and discussed. The study of the substitutional energies indicates that these substitutions are favorable. Comparison between the pure and doped hosts absorption coefficients shows that this deeper band opens up more photon absorption channels and could therefore increase the solar-light absorption with respect to the host.

Interpretación geométrica de redes neuronales recurrentes discretas mediante grafos completos

El objetivo del presente trabajo de investigación es explorar nuevas técnicas de implementación, basadas en grafos, para las Redes de Neuronas, con el fin de simplificar y optimizar las arquitecturas y la complejidad computacional de las mismas. Hemos centrado nuestra atención en una clase de Red de Neuronas: las Redes de Neuronas Recursivas (RNR), también conocidas como redes de Hopfield. El problema de obtener la matriz sináptica asociada con una RNR imponiendo un determinado número de vectores como puntos fijos, no está en absoluto resuelto, el número de vectores prototipo que pueden ser almacenados en la red, cuando se utiliza la ley de Hebb, es bastante limitado, la red se satura rápidamente cuando se pretende almacenar nuevos prototipos. La ley de Hebb necesita, por tanto, ser revisada. Algunas aproximaciones dirigidas a solventar dicho problema, han sido ya desarrolladas. Nosotros hemos desarrollado una nueva aproximación en la forma de implementar una RNR en orden a solucionar estos problemas. La matriz sináptica es obtenida mediante la superposición de las componentes de los vectores prototipo, sobre los vértices de un Grafo, lo cual puede ser también interpretado como una coloración de dicho grafo. Cuando el periodo de entrenamiento se termina, la matriz de adyacencia del Grafo Resultante o matriz de pesos, presenta ciertas propiedades por las cuales dichas matrices serán llamadas tetraédricas. La energía asociada a cualquier estado de la red es representado por un punto (a,b) de R2. Cada uno de los puntos de energía asociados a estados que disten lo mismo del vector cero está localizado sobre la misma línea de energía de R2. El espacio de vectores de estado puede, por tanto, clasificarse en n clases correspondientes a cada una de las n diferentes distancias que puede tener cualquier vector al vector cero. La matriz (n x n) de pesos puede reducirse a un n-vector; de esta forma, tanto el tiempo de computación como el espacio de memoria requerido par almacenar los pesos, son simplificados y optimizados. En la etapa de recuperación, es introducido un vector de parámetros R2, éste es utilizado para controlar la capacidad de la red: probaremos que lo mayor es la componente a¡, lo menor es el número de puntos fijos pertenecientes a la línea de energía R¡. Una vez que la capacidad de la red ha sido controlada mediante este parámetro, introducimos otro parámetro, definido como la desviación del vector de pesos relativos, este parámetro sirve para disminuir ostensiblemente el número de parásitos. A lo largo de todo el trabajo, hemos ido desarrollando un ejemplo, el cual nos ha servido para ir corroborando los resultados teóricos, los algoritmos están escritos en un pseudocódigo, aunque a su vez han sido implamentados utilizando el paquete Mathematica 2.2., mostrándolos en un volumen suplementario al texto.---ABSTRACT---The aim of the present research is intended to explore new specifícation techniques of Neural Networks based on Graphs to be used in the optimization and simplification of Network Architectures and Computational Complexhy. We have focused our attention in a, well known, class of Neural Networks: the Recursive Neural Networks, also known as Hopfield's Neural Networks. The general problem of constructing the synaptic matrix associated with a Recursive Neural Network imposing some vectors as fixed points is fer for completery solved, the number of prototype vectors (learning patterns) which can be stored by Hebb's law is rather limited and the memory will thus quickly reach saturation if new prototypes are continuously acquired in the course of time. Hebb's law needs thus to be revised in order to allow new prototypes to be stored at the expense of the older ones. Some approaches related with this problem has been developed. We have developed a new approach of implementing a Recursive Neural Network in order to sob/e these kind of problems, the synaptic matrix is obtained superposing the components of the prototype vectors over the vértices of a Graph which may be interpreted as a coloring of the Graph. When training is finished the adjacency matrix of the Resulting Graph or matrix of weights presents certain properties for which it may be called a tetrahedral matrix The energy associated to any possible state of the net is represented as a point (a,b) in R2. Every one of the energy points associated with state-vectors having the same Hamming distance to the zero vector are located over the same energy Une in R2. The state-vector space may be then classified in n classes according to the n different possible distances firom any of the state-vectors to the zero vector The (n x n) matrix of weights may also be reduced to a n-vector of weights, in this way the computational time and the memory space required for obtaining the weights is optimized and simplified. In the recall stage, a parameter vectora is introduced, this parameter is used for controlling the capacity of the net: it may be proved that the bigger is the r, component of J, the lower is the number of fixed points located in the r¡ energy line. Once the capacity of the net has been controlled by the ex parameter, we introduced other parameter, obtained as the relative weight vector deviation parameter, in order to reduce the number of spurious states. All along the present text, we have also developed an example, which serves as a prove for the theoretical results, the algorithms are shown in a pseudocode language in the text, these algorithm so as the graphics have been developed also using the Mathematica 2.2. mathematical package which are shown in a supplementary volume of the text.

Photovoltaic application of the V, Cr and Mn-doped cadmium thioindate

The CdIn2S4 spinel semiconductor is a potential photovoltaic material due to its energy band gap and absorption properties. These optoelectronic properties can be potentiality improved by the insertion of intermediate states into the energy bandgap. We explore this possibility using M = Cr, V and Mn as an impurity. We analyze with first-principles almost all substitutions of the host atoms by M at the octahedral and tetrahedral sites in the normal and inverse spinel structures. In almost all cases, the impurities introduce deeper bands into the host energy bandgap. Depending on the site substitution, these bands are full, empty or partially-full. It increases the number of possible inter-band transitions and the possible applications in optoelectronic devices. The contribution of the impurity states to these bands and the substitutional energies indicate that these impurities are energetically favorable for some sites in the host spinel. The absorption coefficients in the independent-particle approximation show that these deeper bands open additional photon absorption channels. It could therefore increase the solar-light absorption with respect to the host.

Influencia de la temperatura en la deflexión de firmes flexibles

En esta investigación se ha estudiado el efecto de la variación de la temperatura en la deflexión de firmes flexibles. En primer lugar se han recopilado los criterios existentes de ajuste de la deflexión por efecto de la temperatura. Posteriormente, se ha llevado a cabo un estudio empírico mediante la auscultación de las deflexiones en cinco tramos de carretera con firme flexible y con diferentes espesores de mezclas bituminosas (entre 10 y 30 cm). Las medidas se han efectuado en dos campañas (verano e invierno), tratando de abarcar un amplio rango de temperaturas. En cada campaña, se han llevado a cabo distintas auscultaciones a diferentes temperaturas. Las medidas de cada campaña se han realizado el mismo día. Se han obtenido los coeficientes empíricos de ajuste por temperatura para cada tramo analizado. Además, se ha realizado un estudio teórico mediante la elaboración de diferentes modelos (multicapa elástico lineal, multicapa visco-elástico lineal y elementos finitos) que reproducen la respuesta estructural de los firmes flexibles auscultados. La caracterización mecánica de las mezclas bituminosas se ha realizado mediante ensayos de módulo complejo en laboratorio, a diferentes temperaturas y frecuencias, sobre testigos extraídos en las carreteras estudiadas. Se han calculado los coeficientes teóricos de ajuste por temperatura para cada modelo elaborado y tramo analizado. Finalmente, se ha realizado un estudio comparativo entre los distintos coeficientes de ajuste (existentes, empíricos y teóricos), que ha puesto de manifiesto que, en todos los casos analizados, los coeficientes obtenidos en el modelo de elementos finitos son los que más se aproximan a los coeficientes empíricos (valor de referencia para los tramos analizados). El modelo desarrollado de elementos finitos permite reproducir el comportamiento visco-elástico de las mezclas bituminosas y el carácter dinámico de las cargas aplicadas. Se han utilizado elementos tipo tetraedro isoparamétrico lineal (C3D8R) para el firme y la parte superior del cimiento, mientras que para la parte inferior se han empleado elementos infinitos (CIN3D8). In this research the effect produced by the temperature change on flexible pavements deflection is analysed. First, the existing criteria of deflection adjustment by temperature were collected. Additionally, an empirical analysis was carried out, consisting on deflection tests in five flexible-pavement road sections with different asphalt mix thickness (from 10 to 30 cm). The measures were taken in two seasons (summer and winter) in an effort to register a wide range of temperatures. Different surveys were carried out at different temperatures in each season. The tests of each season were done at the same day. The empirical temperature adjustment factors for every analysed section were obtained. A theoretical study was carried out by developing different models (linear elastic multilayer, linear visco-elastic multilayer and finite elements) that reproduce the structural response of the tested flexible pavements. The mechanical characterization of the asphalt mixes was achieved through laboratory complex-modulus tests at different temperatures and frequencies, using pavement cores from the surveyed roads. The theoretical temperature adjustment factors for each model developed and each section analysed were calculated. Finally, a comparative study among the different adjustment factors (existing, empirical and theoretical) was carried out. It has shown that, in all analysed cases, the factors obtained with the finite elements model are the closest to the empirical factors (reference value for the analysed sections). The finite elements model developed makes it possible to reproduce the visco-elastic behavior of the asphalt mixes and the dynamic nature of the applied loads. Linear isoparametric tetrahedral elements (C3D8R) have been used for the pavement and the subgrade, while infinite elements (CIN3D8) have been used for the foundations.

Third-generation synchrotron x-ray diffraction of 6-μm crystal of raite, ≈Na3Mn3Ti0.25Si8O20(OH)2⋅10H2O, opens up new chemistry and physics of low-temperature minerals

The crystal structure of raite was solved and refined from data collected at Beamline Insertion Device 13 at the European Synchrotron Radiation Facility, using a 3 × 3 × 65 μm single crystal. The refined lattice constants of the monoclinic unit cell are a = 15.1(1) Å; b = 17.6(1) Å; c = 5.290(4) Å; β = 100.5(2)°; space group C2/m. The structure, including all reflections, refined to a final R = 0.07. Raite occurs in hyperalkaline rocks from the Kola peninsula, Russia. The structure consists of alternating layers of a hexagonal chicken-wire pattern of 6-membered SiO4 rings. Tetrahedral apices of a chain of Si six-rings, parallel to the c-axis, alternate in pointing up and down. Two six-ring Si layers are connected by edge-sharing octahedral bands of Na+ and Mn3+ also parallel to c. The band consists of the alternation of finite Mn–Mn and Na–Mn–Na chains. As a consequence of the misfit between octahedral and tetrahedral elements, regions of the Si–O layers are arched and form one-dimensional channels bounded by 12 Si tetrahedra and 2 Na octahedra. The channels along the short c-axis in raite are filled by isolated Na(OH,H2O)6 octahedra. The distorted octahedrally coordinated Ti4+ also resides in the channel and provides the weak linkage of these isolated Na octahedra and the mixed octahedral tetrahedral framework. Raite is structurally related to intersilite, palygorskite, sepiolite, and amphibole.

Design of potent and selective human cathepsin K inhibitors that span the active site

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

Biochemical evolution III: Polymerization on organophilic silica-rich surfaces, crystal–chemical modeling, formation of first cells, and geological clues

Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources. Crystal–chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite). Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al–OH site per 25 neutral Si tetrahedral sites. Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species. Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms. The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich “soup.” Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments. Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones. All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species. Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Self-assembly of a heteroduplex helicate from two different ligand strands and Cu(II) cations.

Cu(II) ions have been reacted with a 1/1 mixture of two linear ligands, one containing three 2,2'- bipyridine groups and the other three 2,2':6',2"-terpyridine groups. Absorption spectroscopy and fast atom bombardment mass spectrometry indicate the formation of a trinuclear complex containing one ligand of each kind. Determination of the crystal structure of this compound has confirmed that it is indeed a linear trinuclear complex in which two different ligands are wrapped in a helical fashion around the pentacoordinated metal ions. The central coordination geometry is trigonal bipyramidal; the two lateral Cu(II) ions are in a square pyramidal environment. Thus, a heteroduplex helicate is formed by the self-assembly of two different ligand strands and three specific metal ions induced by the coordination number and geometry of the latter. The self-assembly process may be considered to result from the reading of the steric and binding information present in the two ligands by Cu(II) ions through a pentacoordination algorithm. The same ligands have been shown earlier to yield homoduplex helicates from ions of tetrahedral and octahedral coordination geometry and strands of bidentate bipyridines and tridentate terpyridines, respectively. These two types of artificial double helical species may be related on one hand to the natural homoduplex nucleic acids and on the other hand to the DNA:RNA heteroduplex.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.