Broadband Fourier transform rotational spectroscopy for structure determination: The water heptamer

Over the recent years chirped-pulse, Fourier-transform microwave (CP-FTMW) spectrometers have chan- ged the scope of rotational spectroscopy. The broad frequency and large dynamic range make possible structural determinations in molecular systems of increasingly larger size from measurements of heavy atom (13C, 15N, 18O) isotopes recorded in natural abundance in the same spectrum as that of the parent isotopic species. The design of a broadband spectrometer operating in the 2–8 GHz frequency range with further improvements in sensitivity is presented. The current CP-FTMW spectrometer performance is benchmarked in the analyses of the rotational spectrum of the water heptamer, (H2O)7, in both 2– 8 GHz and 6–18 GHz frequency ranges. Two isomers of the water heptamer have been observed in a pulsed supersonic molecular expansion. High level ab initio structural searches were performed to pro- vide plausible low-energy candidates which were directly compared with accurate structures provided from broadband rotational spectra. The full substitution structure of the most stable species has been obtained through the analysis of all possible singly-substituted isotopologues (H218O and HDO), and a least-squares rm(1) geometry of the oxygen framework determined from 16 different isotopic species compares with the calculated O–O equilibrium distances at the 0.01 Å level.

Broadband Fourier transform rotational spectroscopy for structure determination: The water heptamer

Over the recent years chirped-pulse, Fourier-transform microwave (CP-FTMW) spectrometers have changed the scope of rotational spectroscopy. The broad frequency and large dynamic range make possible structural determinations in molecular systems of increasingly larger size from measurements of heavy atom (C-13, N-15, O-18) isotopes recorded in natural abundance in the same spectrum as that of the parent isotopic species. The design of a broadband spectrometer operating in the 2-8 GHz frequency range with further improvements in sensitivity is presented. The current CP-FTMW spectrometer performance is benchmarked in the analyses of the rotational spectrum of the water heptamer, (H2O)(7), in both 2-8 GHz and 6-18 GHz frequency ranges. Two isomers of the water heptamer have been observed in a pulsed supersonic molecular expansion. High level ab initio structural searches were performed to provide plausible low-energy candidates which were directly compared with accurate structures provided from broadband rotational spectra. The full substitution structure of the most stable species has been obtained through the analysis of all possible singly-substituted isotopologues ((H2O)-O-18 and HDO), and a least-squares r(m)((1)) geometry of the oxygen framework determined from 16 different isotopic species compares with the calculated O-O equilibrium distances at the 0.01 angstrom level. (C) 2013 Elsevier B.V. All rights reserved.

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.

Modeling Yield Surfaces of Various Structural Shapes

This study investigates the possibility of custom fitting a widely accepted approximate yield surface equation (Ziemian, 2000) to the theoretical yield surfaces of five different structural shapes, which include wide-flange, solid and hollow rectangular, and solid and hollow circular shapes. To achieve this goal, a theoretically “exact” but overly complex representation of the cross section’s yield surface was initially obtained by using fundamental principles of solid mechanics. A weighted regression analysis was performed with the “exact” yield surface data to obtain the specific coefficients of three terms in the approximate yield surface equation. These coefficients were calculated to determine the “best” yield surface equation for a given cross section geometry. Given that the exact yield surface shall have zero percentage of concavity, this investigation evaluated the resulting coefficient of determination (

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.

Stereoselective synthesis and structure determination of a bicyclo[3.3.2]decapeptide

By analogy to the structural diversity of covalent bond networks between atoms within organic molecules, one can design topologically diverse peptides from mathematical graphs by assigning amino acids to graph nodes and peptide bonds to graph edges. The key is to use diamino acids or amino diacids as equivalents of trivalent graph nodes, which enables a variety of graph topologies beyond the standard linear and monocyclic graphs in natural peptides. Here the bicyclic decapeptide A1FGk2VFPE1AG2 (1b) was prepared and crystallized to assign its bridge stereochemistry. The bridge configuration appears as planned by the chirality of the branching amino acids. Bicyclization furthermore depends on the presence of matched chiralities in the branching amino acids. The stereoselective formation of the second bridge opens the way for the synthesis of a large family of bicyclic peptides as promising new scaffolds for drug design.

Absolute polarity determination of teeth cementum by phase sensitive second harmonic generation microscopy

The absolute sign of local polarity in relation to the biological growth direction has been investigated for teeth cementum using phase sensitive second harmonic generation microscopy (PS-SHGM) and a crystal of 2-cyclooctylamino-5-nitropyridine (COANP) as a nonlinear optic (NLO) reference material. A second harmonic generation (SHG) response was found in two directions of cementum: radial (acellular extrinsic fibers that are oriented more or less perpendicular to the root surface) and circumferential (cellular intrinsic fibers that are oriented more or less parallel to the surface). A mono-polar state was demonstrated for acellular extrinsic cementum. However, along the different parts of cementum in circumferential direction, two corresponding domains were observed featuring an opposite sign of polarity indicative for a bi-polar microscopic state of cellular intrinsic cementum. The phase information showed that the orientation of radial collagen fibrils of cementum is regularly organized with the donor (D) groups pointing to the surface. Circumferential collagen molecules feature orientational disorder and are oriented up and down in random manner showing acceptor or donor groups at the surface of cementum. Considering that the cementum continues to grow in thickness throughout life, we can conclude that the cementum is growing circumferentially in two opposite directions and radially in one direction. A Markov chain type model for polarity formation in the direction of growth predicts D-groups preferably appearing at the fiber front.

Experimental determination of the eutectic temperature in air of the CuO-TiO2 pseudobinary system

Eutectic temperature and composition in the CuO–TiO2 pseudobinary system have been experimentally determined in air by means differential thermal analysis (DTA), thermogravimetry (TG) and hot-stage microscopy (HSM). Samples of the new eutectic composition treated at different temperatures have been characterized by X-ray diffraction (XRD) and X-ray absorption near-edge structural spectroscopy (XANES) to identify phases and to determine the Cu valence state, respectively. The results show that the eutectic temperature in air is higher by 100 °C (∼1000 °C) for a Ti-richer composition (XTiO2=25 mol%) than the one calculated in the literature. The reduction of Cu2+ to Cu+ takes places at about 1030 °C. The existence of Cu2TiO3 and Cu3TiO4 has been confirmed by XRD in the temperature range between 1045 and 1200 °C.

Open-atmosphere structural depth profiling of multilayer samples of photovoltaic interest using laser-induced plasma spectrometry

The present work aims to assess Laser-Induced Plasma Spectrometry (LIPS) as a tool for the characterization of photovoltaic materials. Despite being a well-established technique with applications to many scientific and industrial fields, so far LIPS is little known to the photovoltaic scientific community. The technique allows the rapid characterization of layered samples without sample preparation, in open atmosphere and in real time. In this paper, we assess LIPS ability for the determination of elements that are difficult to analyze by other broadly used techniques, or for producing analytical information from very low-concentration elements. The results of the LIPS characterization of two different samples are presented: 1) a 90 nm, Al-doped ZnO layer deposited on a Si substrate by RF sputtering and 2) a Te-doped GaInP layer grown on GaAs by Metalorganic Vapor Phase Epitaxy. For both cases, the depth profile of the constituent and dopant elements is reported along with details of the experimental setup and the optimization of key parameters. It is remarkable that the longest time of analysis was ∼10 s, what, in conjunction with the other characteristics mentioned, makes of LIPS an appealing technique for rapid screening or quality control whether at the lab or at the production line.

Determination of the crystal and magnetic structure of the DyCrO4-scheelite polymorph by neutron diffraction

Neutron diffraction data of DyCrO4 oxide, prepared at 4 GPa and 833 K from the ambient pressure zircon-type, reveal that crystallize with the scheelite-type structure, space group I41/a. Accompanying this structural phase transition induced by pressure the magnetic properties change dramatically from ferromagnetism in the case of zircon to antiferromagnetism for the scheelite polymorph with a T N= 19 K. The analysis of the neutron diffraction data obtained at 1.2 K has been used to determine the magnetic structure of this DyCrO4-scheelite oxide which can be described with a k = [0, 0, 0] as propagation vector, where the Dy and Cr moments are lying in the ab-plane of the scheelite structure. The ordered magnetic moments are 10 µB and 1 µB for Dy+3 and Cr+5 respectively

Structural efficiency variation with the problem size in some bending problems = Variación del rendimiento estructural con el tamaño del problema en algunos casos de flexión simple

Existe normalmente el propósito de obtener la mejor solución posible cuando se plantea un problema estructural, entendiendo como mejor la solución que cumpliendo los requisitos estructurales, de uso, etc., tiene un coste físico menor. En una primera aproximación se puede representar el coste físico por medio del peso propio de la estructura, lo que permite plantear la búsqueda de la mejor solución como la de menor peso. Desde un punto de vista práctico, la obtención de buenas soluciones—es decir, soluciones cuyo coste sea solo ligeramente mayor que el de la mejor solución— es una tarea tan importante como la obtención de óptimos absolutos, algo en general difícilmente abordable. Para disponer de una medida de la eficiencia que haga posible la comparación entre soluciones se propone la siguiente definición de rendimiento estructural: la razón entre la carga útil que hay que soportar y la carga total que hay que contabilizar (la suma de la carga útil y el peso propio). La forma estructural puede considerarse compuesta por cuatro conceptos, que junto con el material, definen una estructura: tamaño, esquema, proporción, y grueso.Galileo (1638) propuso la existencia de un tamaño insuperable para cada problema estructural— el tamaño para el que el peso propio agota una estructura para un esquema y proporción dados—. Dicho tamaño, o alcance estructural, será distinto para cada material utilizado; la única información necesaria del material para su determinación es la razón entre su resistencia y su peso especifico, una magnitud a la que denominamos alcance del material. En estructuras de tamaño muy pequeño en relación con su alcance estructural la anterior definición de rendimiento es inútil. En este caso —estructuras de “talla nula” en las que el peso propio es despreciable frente a la carga útil— se propone como medida del coste la magnitud adimensional que denominamos número de Michell, que se deriva de la “cantidad” introducida por A. G. M. Michell en su artículo seminal de 1904, desarrollado a partir de un lema de J. C. Maxwell de 1870. A finales del siglo pasado, R. Aroca combino las teorías de Galileo y de Maxwell y Michell, proponiendo una regla de diseño de fácil aplicación (regla GA), que permite la estimación del alcance y del rendimiento de una forma estructural. En el presente trabajo se estudia la eficiencia de estructuras trianguladas en problemas estructurales de flexión, teniendo en cuenta la influencia del tamaño. Por un lado, en el caso de estructuras de tamaño nulo se exploran esquemas cercanos al optimo mediante diversos métodos de minoración, con el objetivo de obtener formas cuyo coste (medido con su numero deMichell) sea muy próximo al del optimo absoluto pero obteniendo una reducción importante de su complejidad. Por otro lado, se presenta un método para determinar el alcance estructural de estructuras trianguladas (teniendo en cuenta el efecto local de las flexiones en los elementos de dichas estructuras), comparando su resultado con el obtenido al aplicar la regla GA, mostrando las condiciones en las que es de aplicación. Por último se identifican las líneas de investigación futura: la medida de la complejidad; la contabilidad del coste de las cimentaciones y la extensión de los métodos de minoración cuando se tiene en cuenta el peso propio. ABSTRACT When a structural problem is posed, the intention is usually to obtain the best solution, understanding this as the solution that fulfilling the different requirements: structural, use, etc., has the lowest physical cost. In a first approximation, the physical cost can be represented by the self-weight of the structure; this allows to consider the search of the best solution as the one with the lowest self-weight. But, from a practical point of view, obtaining good solutions—i.e. solutions with higher although comparable physical cost than the optimum— can be as important as finding the optimal ones, because this is, generally, a not affordable task. In order to have a measure of the efficiency that allows the comparison between different solutions, a definition of structural efficiency is proposed: the ratio between the useful load and the total load —i.e. the useful load plus the self-weight resulting of the structural sizing—. The structural form can be considered to be formed by four concepts, which together with its material, completely define a particular structure. These are: Size, Schema, Slenderness or Proportion, and Thickness. Galileo (1638) postulated the existence of an insurmountable size for structural problems—the size for which a structure with a given schema and a given slenderness, is only able to resist its self-weight—. Such size, or structural scope will be different for every different used material; the only needed information about the material to determine such size is the ratio between its allowable stress and its specific weight: a characteristic length that we name material structural scope. The definition of efficiency given above is not useful for structures that have a small size in comparison with the insurmountable size. In this case—structures with null size, inwhich the self-weight is negligible in comparisonwith the useful load—we use as measure of the cost the dimensionless magnitude that we call Michell’s number, an amount derived from the “quantity” introduced by A. G. M. Michell in his seminal article published in 1904, developed out of a result from J. C.Maxwell of 1870. R. Aroca joined the theories of Galileo and the theories of Maxwell and Michell, obtaining some design rules of direct application (that we denominate “GA rule”), that allow the estimation of the structural scope and the efficiency of a structural schema. In this work the efficiency of truss-like structures resolving bending problems is studied, taking into consideration the influence of the size. On the one hand, in the case of structures with null size, near-optimal layouts are explored using several minimization methods, in order to obtain forms with cost near to the absolute optimum but with a significant reduction of the complexity. On the other hand, a method for the determination of the insurmountable size for truss-like structures is shown, having into account local bending effects. The results are checked with the GA rule, showing the conditions in which it is applicable. Finally, some directions for future research are proposed: the measure of the complexity, the cost of foundations and the extension of optimization methods having into account the self-weight.

NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains

Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ1I. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its Ka value is 0.20 × 106 M−1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.