Modelling of chloride penetration into non-saturated concrete: Case study application for real marine off shore structures

The aim of this paper is to explain the chloride concentration profiles obtained experimentally from control samples of an offshore platform after 25 years of service life. The platform is located 12 km off the coast of the Brazilian province Rio Grande do Norte, in the north-east of Brazil. The samples were extracted at different orientations and heights above mean sea level. A simple model based on Fick’s second law is considered and compared with a finite element model which takes into account transport of chloride ions by diffusion and convection. Results show that convective flows significantly affect the studied chloride penetrations. The convection velocity is obtained by fitting the finite element solution to the experimental data and seems to be directly proportional to the height above mean sea level and also seems to depend on the orientation of the face of the platform. This work shows that considering solely diffusion as transport mechanism does not allow a good prediction of the chloride profiles. Accounting for capillary suction due to moisture gradients permits a better interpretation of the material’s behaviour.

Continuum models of the mechanical behavior of rolled and die-cast magnesium alloys

En los últimos años ha habido una fuerte tendencia a disminuir las emisiones de CO2 y su negativo impacto medioambiental. En la industria del transporte, reducir el peso de los vehículos aparece como la mejor opción para alcanzar este objetivo. Las aleaciones de Mg constituyen un material con gran potencial para el ahorro de peso. Durante la última década se han realizado muchos esfuerzos encaminados a entender los mecanismos de deformación que gobiernan la plasticidad de estos materiales y así, las aleaciones de Mg de colada inyectadas a alta presión y forjadas son todavía objeto de intensas campañas de investigación. Es ahora necesario desarrollar modelos que contemplen la complejidad inherente de los procesos de deformación de éstos. Esta tesis doctoral constituye un intento de entender mejor la relación entre la microestructura y el comportamiento mecánico de aleaciones de Mg, y dará como resultado modelos de policristales capaces de predecir propiedades macro- y microscópicas. La deformación plástica de las aleaciones de Mg está gobernada por una combinación de mecanismos de deformación característicos de la estructura cristalina hexagonal, que incluye el deslizamiento cristalográfico en planos basales, prismáticos y piramidales, así como el maclado. Las aleaciones de Mg de forja presentan texturas fuertes y por tanto los mecanismos de deformación activos dependen de la orientación de la carga aplicada. En este trabajo se ha desarrollado un modelo de plasticidad cristalina por elementos finitos con el objetivo de entender el comportamiento macro- y micromecánico de la aleación de Mg laminada AZ31 (Mg-3wt.%Al-1wt.%Zn). Este modelo, que incorpora el maclado y tiene en cuenta el endurecimiento por deformación debido a las interacciones dislocación-dislocación, dislocación-macla y macla-macla, predice exitosamente las actividades de los distintos mecanismos de deformación y la evolución de la textura con la deformación. Además, se ha llevado a cabo un estudio que combina difracción de electrones retrodispersados en tres dimensiones y modelización para investigar el efecto de los límites de grano en la propagación del maclado en el mismo material. Ambos, experimentos y simulaciones, confirman que el ángulo de desorientación tiene una influencia decisiva en la propagación del maclado. Se ha observado que los efectos no-Schmid, esto es, eventos de deformación plástica que no cumplen la ley de Schmid con respecto a la carga aplicada, no tienen lugar en la vecindad de los límites de baja desorientación y se hacen más frecuentes a medida que la desorientación aumenta. Esta investigación también prueba que la morfología de las maclas está altamente influenciada por su factor de Schmid. Es conocido que los procesos de colada suelen dar lugar a la formación de microestructuras con una microporosidad elevada, lo cuál afecta negativamente a sus propiedades mecánicas. La aplicación de presión hidrostática después de la colada puede reducir la porosidad y mejorar las propiedades aunque es poco conocido su efecto en el tamaño y morfología de los poros. En este trabajo se ha utilizado un enfoque mixto experimentalcomputacional, basado en tomografía de rayos X, análisis de imagen y análisis por elementos finitos, para la determinación de la distribución tridimensional (3D) de la porosidad y de la evolución de ésta con la presión hidrostática en la aleación de Mg AZ91 (Mg- 9wt.%Al-1wt.%Zn) colada por inyección a alta presión. La distribución real de los poros en 3D obtenida por tomografía se utilizó como input para las simulaciones por elementos finitos. Los resultados revelan que la aplicación de presión tiene una influencia significativa tanto en el cambio de volumen como en el cambio de forma de los poros que han sido cuantificados con precisión. Se ha observado que la reducción del tamaño de éstos está íntimamente ligada con su volumen inicial. En conclusión, el modelo de plasticidad cristalina propuesto en este trabajo describe con éxito los mecanismos intrínsecos de la deformación de las aleaciones de Mg a escalas meso- y microscópica. Más especificamente, es capaz de capturar las activadades del deslizamiento cristalográfico y maclado, sus interacciones, así como los efectos en la porosidad derivados de los procesos de colada. ---ABSTRACT--- The last few years have seen a growing effort to reduce CO2 emissions and their negative environmental impact. In the transport industry more specifically, vehicle weight reduction appears as the most straightforward option to achieve this objective. To this end, Mg alloys constitute a significant weight saving material alternative. Many efforts have been devoted over the last decade to understand the main mechanisms governing the plasticity of these materials and, despite being already widely used, high pressure die-casting and wrought Mg alloys are still the subject of intense research campaigns. Developing models that can contemplate the complexity inherent to the deformation of Mg alloys is now timely. This PhD thesis constitutes an attempt to better understand the relationship between the microstructure and the mechanical behavior of Mg alloys, as it will result in the design of polycrystalline models that successfully predict macro- and microscopic properties. Plastic deformation of Mg alloys is driven by a combination of deformation mechanisms specific to their hexagonal crystal structure, namely, basal, prismatic and pyramidal dislocation slip as well as twinning. Wrought Mg alloys present strong textures and thus specific deformation mechanisms are preferentially activated depending on the orientation of the applied load. In this work a crystal plasticity finite element model has been developed in order to understand the macro- and micromechanical behavior of a rolled Mg AZ31 alloy (Mg-3wt.%Al-1wt.%Zn). The model includes twinning and accounts for slip-slip, slip-twin and twin-twin hardening interactions. Upon calibration and validation against experiments, the model successfully predicts the activity of the various deformation mechanisms and the evolution of the texture at different deformation stages. Furthermore, a combined three-dimensional electron backscatter diffraction and modeling approach has been adopted to investigate the effect of grain boundaries on twin propagation in the same material. Both experiments and simulations confirm that the misorientation angle has a critical influence on twin propagation. Non-Schmid effects, i.e. plastic deformation events that do not comply with the Schmid law with respect to the applied stress, are absent in the vicinity of low misorientation boundaries and become more abundant as misorientation angle increases. This research also proves that twin morphology is highly influenced by the Schmid factor. Finally, casting processes usually lead to the formation of significant amounts of gas and shrinkage microporosity, which adversely affect the mechanical properties. The application of hydrostatic pressure after casting can reduce the porosity and improve the properties but little is known about the effects on the casting’s pores size and morphology. In this work, an experimental-computational approach based on X-ray computed tomography, image analysis and finite element analysis is utilized for the determination of the 3D porosity distribution and its evolution with hydrostatic pressure in a high pressure diecast Mg AZ91 alloy (Mg-9wt.%Al-1wt.%Zn). The real 3D pore distribution obtained by tomography is used as input for the finite element simulations using an isotropic hardening law. The model is calibrated and validated against experimental stress-strain curves. The results reveal that the pressure treatment has a significant influence both on the volume and shape changes of individuals pores, which have been precisely quantified, and which are found to be related to the initial pore volume. In conclusion, the crystal plasticity model proposed in this work successfully describes the intrinsic deformation mechanisms of Mg alloys both at the mesoscale and the microscale. More specifically, it can capture slip and twin activities, their interactions, as well as the potential porosity effects arising from casting processes.

Urbanism and dictatorship: perspectives of the field of urban studies

In 1933 public letter to Wilhelm Furtwängler, Joseph Goebbels synthesized the official understanding of the link between politics, art and society in the early steps of the Third Reich. By assuming the ethos of art, politics acquired a plastic agency to mold its objects —population and the state— as a unified entity in the form of a ‘national-popular community’ (Volksgemeinschaft); in turn, by infusing art with a political valence, it became part of a wider governmental apparatus that reshaped aesthetic discourses and practices. Similar remarks could be made about the ordering of cities and territories in this period. Dictatorial imaginations mobilized urbanism —including urban theory, urban design and planning— as a fundamental tool for social organization. Under their aegis the production of space became a moment in a wider production of society. Many authors suggest that this political-spatial nexus is intrinsic to modernity itself, beyond dictatorial regimes. In this light, I propose to use dictatorial urbanisms as an analytical opportunity to delve into some concealed features of modern urban design and planning. This chapter explores some of these aspects from a theoretical standpoint, focusing on the development of dictatorial planning mentalities and spatial rationalities and drawing links to other historical episodes in order to inscribe the former in a broader genealogy of urbanism. Needless to say, I don’t suggest that we use dictatorships as mere templates to understand modern productions of space. Instead, these cases provide a crude version of some fundamental drives in the operationalization of urbanism as an instrument of social regulation, showing how far the modern imagination of sociospatial orderings can go. Dictatorial urbanisms constituted a set of experiences where many dreams and aspirations of modern planning went to die. But not, as the conventional account would have it, because the former were the antithesis of the latter, but rather because they worked as the excess of a particular orientation of modern spatial governmentalities — namely, their focus on calculation, social engineering and disciplinary spatialities, and their attempt to subsume a wide range of everyday practices under institutional structuration by means of spatial mediations. In my opinion the interest of dictatorial urbanisms lies in their role as key regulatory episodes in a longer history of our urban present. They stand as a threshold between the advent of planning in the late 19th and early 20th century, and its final consolidation as a crucial state instrument after World War II. We need, therefore, to pay attention to these experiences vis-à-vis the alleged ‘normal’ development of the field in contemporary democratic countries in order to develop a full comprehension thereof.

Modelling of chloride penetration into non-saturated concrete: case study application for real marine offshore structures

The aim of this paper is to explain the chloride concentration profiles obtained experimentally from control samples of an offshore platform after 25 years of service life. The platform is located 12 km off the coast of the Brazilian province Rio Grande do Norte, in the north-east of Brazil. The samples were extracted at different orientations and heights above mean sea level. A simple model based on Fick’s second law is considered and compared with a finite element model which takes into account transport of chloride ions by diffusion and convection. Results show that convective flows significantly affect the studied chloride penetrations. The convection velocity is obtained by fitting the finite element solution to the experimental data and seems to be directly proportional to the height above mean sea level and also seems to depend on the orientation of the face of the platform. This work shows that considering solely diffusion as transport mechanism does not allow a good prediction of the chloride profiles. Accounting for capillary suction due to moisture gradients permits a better interpretation of the material’s behaviour

What is the optimal stitching orientation from an aerial survey perspective?

Mosaicing is a technique that allows obtaining a large high resolution image by stitching several images together. These base images are usually acquired from an elevated point of view. Until recently, low-altitude image acquisition has been performed typically by using using airplanes, as well as other manned platforms. However, mini unmanned aerial vehicles (MUAV) endowed with a camera have lately made this task more available for small for cicil applications, for example for small farmers in order to obtain accurate agronomic information about their crop fields. The stitching orientation, or the image acquisition orientation usually coincides with the aircraft heading assuming a downwards orientation of the camera. In this paper, the efect of the image orientation in the eficiency of the aerial coverage path planning is studied. Moreover, an algorithm to compute an optimal stitching orientation angle is proposed and results are numerically compared with classical approaches.

Using the Cross-Entropy Method for Control Optimization: A Case Study of See-and-Avoid on Unmanned Aerial Vehicles

This paper presents an adaptation of the Cross-Entropy (CE) method to optimize fuzzy logic controllers. The CE is a recently developed optimization method based on a general Monte-Carlo approach to combinatorial and continuous multi-extremal optimization and importance sampling. This work shows the application of this optimization method to optimize the inputs gains, the location and size of the different membership functions' sets of each variable, as well as the weight of each rule from the rule's base of a fuzzy logic controller (FLC). The control system approach presented in this work was designed to command the orientation of an unmanned aerial vehicle (UAV) to modify its trajectory for avoiding collisions. An onboard looking forward camera was used to sense the environment of the UAV. The information extracted by the image processing algorithm is the only input of the fuzzy control approach to avoid the collision with a predefined object. Real tests with a quadrotor have been done to corroborate the improved behavior of the optimized controllers at different stages of the optimization process.

Polymer interaction with macroscopic carbon nanotube fibres and fabrication of nanostructured composites

El ensamblado de nanotubos de carbono (CNT) como una fibra macroscópica en la cual están orientados preferentemente paralelos entre sí y al eje de la fibra, ha dado como resultado un nuevo tipo de fibra de altas prestaciones derivadas de la explotación eficiente de las propiedades axiales de los CNTs, y que tiene un gran número de aplicaciones potenciales. Fibras continuas de CNTs se produjeron en el Instituto IMDEA Materiales mediante el proceso de hilado directo durante la reacción de síntesis por deposición química de vapores. Uno de los objetivos de esta tesis es el estudio de la estructura de estas fibras mediante técnicas del estado del arte de difracción de rayos X de sincrotrón y la elaboración de un modelo estructural de dicho material. Mediciones texturales de adsorción de gases, análisis de micrografías de electrones y dispersión de rayos X de ángulo alto y bajo (WAXS/SAXS) indican que el material tiene una estructura mesoporosa con una distribución de tamaño de poros ancha derivada del amplio rango de separaciones entre manojos de CNTs, así como una superficie específica de 170m2/g. Los valores de dimensión fractal obtenidos mediante SAXS y análisis Barrett-Joyner-Halenda (BJH) de mediciones texturales coinciden en 2.4 y 2.5, respectivamente, resaltando el carácter de red de la estructura de dichas fibras. La estructura mesoporosa y tipo hilo de las fibra de CNT es accesible a la infiltración de moléculas externas (líquidos o polímeros). En este trabajo se estudian los cambios en la estructura multiescala de las fibras de CNTs al interactuar con líquidos y polímeros. Los efectos de la densificación en la estructura de fibras secas de CNT son estudiados mediante WAXS/SAXS. El tratamiento de densificación junta los manojos de la fibra (los poros disminuyen de tamaño), resultando en un incremento de la densidad de la fibra. Sin embargo, los dominios estructurales correspondientes a la transferencia de esfuerzo mecánica y carga eléctrica en los nanotubos no son afectados durante este proceso de densificación; como consecuencia no se produce un efecto sustancial en las propiedades mecánicas y eléctricas. Mediciones de SAXS and fibra de CNT antes y después de infiltración de líquidos confirman la penetración de una gran cantidad de líquidos que llena los poros internos de la fibra pero no se intercalan entre capas de nanotubos adyacentes. La infiltración de cadenas poliméricas de bajo peso molecular tiende a expandir los manojos en la fibra e incrementar el ángulo de apertura de los poros. Los resultados de SAXS indican que la estructura interna de la fibra en términos de la organización de las capas de tubos y su orientación no es afectada cuando las muestras consisten en fibras infiltradas con polímeros de alto peso molecular. La cristalización de varios polímeros semicristalinos es acelerada por la presencia de fibras de CNTs alineados y produce el crecimiento de una capa transcristalina normal a la superficie de la fibra. Esto es observado directamente mediante microscopía óptica polarizada, y detectado mediante calorimetría DSC. Las lamelas en la capa transcristalina tienen orientación de la cadena polimérica paralela a la fibra y por lo tanto a los nanotubos, de acuerdo con los patrones de WAXS. Esta orientación preferencial se sugiere como parte de la fuerza impulsora en la nucleación. La nucleación del dominio cristalino polimérico en la superficie de los CNT no es epitaxial. Ocurre sin haber correspondencia entre las estructuras cristalinas del polímero y los nanotubos. Estas observaciones contribuyen a la compresión del fenómeno de nucleación en CNTs y otros nanocarbonos, y sientan las bases para el desarrollo de composites poliméricos de gran escala basados en fibra larga de CNTs alineados. ABSTRACT The assembly of carbon nanotubes into a macroscopic fibre material where they are preferentially aligned parallel to each other and to the fibre axis has resulted in a new class of high-performance fibres, which efficiently exploits the axial properties of the building blocks and has numerous applications. Long, continuous CNT fibres were produced in IMDEA Materials Institute by direct fibre spinning from a chemical vapour deposition reaction. These fibres have a complex hierarchical structure covering multiple length scales. One objective of this thesis is to reveal this structure by means of state-of-the-art techniques such as synchrotron X-ray diffraction, and to build a model to link the fibre structural elements. Texture and gas absorption measurements, using electron microscopy, wide angle and small angle X-ray scattering (WAXS/SAXS), and pore size distribution analysis by Barrett-Joyner-Halenda (BJH), indicate that the material has a mesoporous structure with a wide pore size distribution arising from the range of fibre bundle separation, and a high surface area _170m2/g. Fractal dimension values of 2.4_2.5 obtained from the SAXS and BJH measurements highlight the network structure of the fibre. Mesoporous and yarn-like structure of CNT fibres make them accessible to the infiltration of foreign molecules (liquid or polymer). This work studies multiscale structural changes when CNT fibres interact with liquids and polymers. The effects of densification on the structure of dry CNT fibres were measured by WAXS/SAXS. The densification treatment brings the fibre bundles closer (pores become smaller), leading to an increase in fibre density. However, structural domains made of the load and charge carrying nanotubes are not affected; consequently, it has no substantial effect on mechanical and electrical properties. SAXS measurements on the CNT fibres before and after liquid infiltration imply that most liquids are able to fill the internal pores but not to intercalate between nanotubes. Successful infiltration of low molecular weight polymer chains tends to expand the fibre bundles and increases the pore-opening angle. SAXS results indicate that the inner structure of the fibre, in terms of the nanotube layer arrangement and the fibre alignment, are not largely affected when infiltrated with polymers of relatively high molecular weight. The crystallisation of a variety of semicrystalline polymers is accelerated by the presence of aligned fibres of CNTs and results in the growth of a transcrystalline layer perpendicular to the fibre surface. This can be observed directly under polarised optical microscope, and detected by the exothermic peaks during differential scanning calorimetry. The discussion on the driving forces for the enhanced nucleation points out the preferential chain orientation of polymer lamella with the chain axis parallel to the fibre and thus to the nanotubes, which is confirmed by two-dimensional WAXS patterns. A non-epitaxial polymer crystal growth habit at the CNT-polymer interface is proposed, which is independent of lattice matching between the polymer and nanotubes. These findings contribute to the discussion on polymer nucleation on CNTs and other nanocarbons, and their implication for the development of large polymer composites based on long and aligned fibres of CNTs.

Stability analysis of a free falling pararotor

The pararotor is a decelerator device based on the autorotation of a rotating wing. When it is dropped, it generates an aerodynamic force parallel to the main motion direction, acting as a decelerating force. In this paper, the rotational motion equations are shown for the vertical flight without any lateral wind component and some simplifying assumptions are introduced to obtain analytic solutions of the motion. First, the equilibrium state is obtained as a function of the main parameters. Then the equilibrium stability is analyzed. The motion stability depends on two nondimensional parameters, which contain geometric, inertia, and aerodynamic characteristics of the device. Based on these two parameters a stability diagram can be defined. Some stability regions with different types of stability trajectories (nodes, spirals, focuses) can be identified for spinning motion around axes close to the major, minor, and intermediate principal axes. It is found that the blades contribute to stability in a case of spin around the intermediate principal inertia axis, which is otherwise unstable. Subsequently, the equations for determining the angles of nutation and spin of the body are obtained, thus defining the orientation of the body for a stationary motion and the parameters on which that position depends.

The peculiar electrical response of liquid crystal-carbon nanotube systems as seen by impedance spectroscopy

Conductive nanoparticles, especially elongated ones such as carbon nanotubes, dramatically modify the electrical behavior of liquid crystal cells. These nanoparticles are known to reorient with liquid crystals in electric fields, causing significant variations of conductivity at minute concentrations of tens or hundreds ppm. The above notwithstanding, impedance spectroscopy of doped cells in the frequency range customarily employed by liquid crystal devices, 100 Hz?10 kHz, shows a relatively simple resistor/capacitor response where the components of the cell can be univocally assigned to single components of the electrical equivalent circuit. However, widening the frequency range up to 1 MHz or beyond reveals a complex behavior that cannot be explained with the same simple EEC. Moreover, the system impedance varies with the application of electric fields, their effect remaining after removing the field. Carbon nanotubes are reoriented together with liquid crystal reorientation when applying voltage, but barely reoriented back upon liquid crystal relaxation once the voltage is removed. Results demonstrate a remarkable variation in the impedance of the dielectric blend formed by liquid crystal and carbon nanotubes, the irreversible orientation of the carbon nanotubes and possible permanent contacts between electrodes.

Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Self-assembly of fibronectin into fibrillar networks underneath dipalmitoyl phosphatidylcholine monolayers: Role of lipid matrix and tensile forces

The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized in situ by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways.

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos–Jun–AP-1 complex may contribute to its functional versatility at different promoters.