Characterizing the microenvironment surrounding protein sites.

Sites are microenvironments within a biomolecular structure, distinguished by their structural or functional role. A site can be defined by a three-dimensional location and a local neighborhood around this location in which the structure or function exists. We have developed a computer system to facilitate structural analysis (both qualitative and quantitative) of biomolecular sites. Our system automatically examines the spatial distributions of biophysical and biochemical properties, and reports those regions within a site where the distribution of these properties differs significantly from control nonsites. The properties range from simple atom-based characteristics such as charge to polypeptide-based characteristics such as type of secondary structure. Our analysis of sites uses non-sites as controls, providing a baseline for the quantitative assessment of the significance of the features that are uncovered. In this paper, we use radial distributions of properties to study three well-known sites (the binding sites for calcium, the milieu of disulfide bridges, and the serine protease active site). We demonstrate that the system automatically finds many of the previously described features of these sites and augments these features with some new details. In some cases, we cannot confirm the statistical significance of previously reported features. Our results demonstrate that analysis of protein structure is sensitive to assumptions about background distributions, and that these distributions should be considered explicitly during structural analyses.     

Quantum chemical studies of protein structure

Quantum chemical methods now permit the prediction of many spectroscopic observables in proteins and related model systems, in addition to electrostatic properties, which are found to be in excellent accord with those determined from experiment. I discuss the developments over the past decade in these areas, including predictions of nuclear magnetic resonance chemical shifts, chemical shielding tensors, scalar couplings and hyperfine (contact) shifts, the isomer shifts and quadrupole splittings in M?ssbauer spectroscopy, molecular energies and conformations, as well as a range of electrostatic properties, such as charge densities, the curvatures, Laplacians and Hessians of the charge density, electrostatic potentials, electric field gradients and electrostatic field effects. The availability of structure/spectroscopic correlations from quantum chemistry provides a basis for using numerous spectroscopic observables in determining aspects of protein structure, in determining electrostatic properties which are not readily accessible from experiment, as well as giving additional confidence in the use of these techniques to investigate questions about chemical bonding and chemical reactions.     

Three-dimensional biochemical patterning of click-based composite hydrogels via thiolene photopolymerization

Hydrogels formed from (meth)acrylated poly(ethylene glycol) precursors are commonly used in a variety of biomedical applications ranging from tissue engineering to biosensors. While this approach has proven quite diverse, a major limitation to this approach is the heterogeneities and nonidealities that arise in the gels from the chain polymerization process, which increases the difficulty in relating the network structure to the final physical properties of the gel. Here we have exploited the specificity and fidelity of the [3+2] cycloaddition reaction to synthesize hydrogels with controlled architectures and improved mechanical properties. Moreover, we demonstrate a general approach toward the integration of multifunctional photoreactive polypeptide sequences into the network structure that provides a facile way to independently tune the 3D chemical and physical properties of the gel. Standard photolithographic techniques were used to generate a variety of two- and three-dimensional patterns as well as controlled biochemical gradients within existing preformed hydrogels.     

Getting to the inside of cells using metabolic control analysis

Metabolic control analysis can relate control properties of an intact system to kinetic properties (elasticity coefficients) of the enzymes within that system. The method formulating the former as matrix inverse of the latter is elaborated here for the general case and founded in standard metabolic control theory. Then a method is developed that accomplishes the reverse: it is shown that a matrix containing all elasticity coefficients and information concerning the pathway structure equals the inverse of a matrix containing flux and concentration control coefficients. As a consequence, by measuring the control properties of an intact system, one is able to deduce its in situ pathway structure and enzyme kinetic properties: This solves the ever-present question of whether the kinetic properties of enzymes in their isolated state differ from those under the conditions prevailing in the cell.     

Natural cellulose fibers from switchgrass with tensile properties similar to cotton and linen

We report the production and characteristics of natural cellulose fibers obtained from the leaves and stems of switchgrass. In this paper, the composition, structure and properties of fibers obtained from the leaves and stem of switchgrass have been studied in comparison to the common natural cellulose fibers, such as cotton, linen and kenaf. The leaves and stems of switchgrass have tensile properties intriguingly similar to that of linen and cotton, respectively. Fibers were obtained from the leaves and stems of switchgrass using a simple alkaline extraction and the structure and properties of the fibers were studied. Fibers obtained from switchgrass leaves have crystallinity of 51%, breaking tenacity of 5.5 g per denier (715 MPa) and breaking elongation of 2.2% whereas the corresponding values for fibers obtained from switchgrass stems are 46%, 2.7 g per denier and 6.8%, respectively. Switchgrass is a relatively easy to grow and high yield biomass crop that can be source to partially substitute the natural and synthetic fibers currently in use. We hope that this research will stimulate interests in using switchgrass as a novel fiber crop in addition to being promoted as a potential source for biofuels.     

Structural and dynamical properties of a full-length HIV-1 integrase: molecular dynamics simulations

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg2+ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glu152; (iii) distances and solvation of the Mg2+; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.     

Isolation and characterization of a second molybdopterin dinucleotide: molybdopterin cytosine dinucleotide

The pterin cofactor (bactopterin) in the molybdoenzyme CO dehydrogenase isolated from Pseudomonas carboxydoflava has previously been shown to differ from molybdopterin in molecular mass, phosphate content, stability, and other properties, implying a novel structure. The structure of the CO dehydrogenase pterin has been investigated in the present studies by alkylation and isolation of the carboxamidomethyl derivative. The alkylated pterin was identified as [di-(carboxamidomethyl)]molybdopterin cytosine dinucleotide on the basis of its absorption properties and by degradation with nucleotide pyrophosphatase yielding carboxamidomethylmolybdopterin and CMP. Further treatment of these products with alkaline phosphatase produced species with absorption and chromatographic properties identical to those of the corresponding dephospho compounds. Molybdopterin cytosine dinucleotide is the second molybdopterin variant to be structurally characterized. The fact that molybdopterin cytosine dinucleotide and molybdopterin guanine dinucleotide contain molybdopterin in their structure shows that the pterin moiety, with its unique dithiolene-containing sidechain, is a structural element which is common to the organic portion of the molybdenum cofactors of many molybdoenzymes.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

Studies on titin PEVK peptides and their interaction

Experiments were conducted on several synthetic and expressed peptides from the PEVK region of titin, the giant muscle protein. Different secondary structure prediction methods based on amino acid sequence gave estimates ranging from over 70% alpha helical to no helix (totally disordered) for the polyE peptide corresponding to human exon 115. Circular dichroism (CD) experiments demonstrated that both the positively charged PPAK modules and the negatively charged PolyE repeats had similar spectral properties with disordered secondary structure predominating. Gel permeation chromatography showed that both PPAK and polyE peptides had 2-4 times larger Stokes radii than expected from their molecular mass. Mixtures of the oppositely charged titin peptides caused no change in apparent secondary structure as observed by circular dichroism or migration properties using native gel electrophoresis. Similarly addition of calcium did not alter the CD spectra or peptide electrophoretic mobility of the individual peptides or their mixtures. The properties of both the PPAK and polyE type peptides suggest that both had most of the characteristic properties to be classified as intrinsically disordered proteins.     

Tritium diagnostics in a fusion reactor

Methods for controlling tritium in a fusion reactor are reviewed. The characteristic features of the interaction of tritium with surfaces are considered, as well as its diffusive properties and its levels and saturation depths for a number of radioactive materials. New properties of boundary layers and new diffusive properties in the polycrystalline structure of metals are revealed. Unique measurements of the diffusive properties of tritium in stainless steel at room temperature are carried out. It is found that the main contribution to the diffusion process comes from the intergranular tritium flux. The capture of tritium by metals in the ITER device is calculated.     

In silico identification of functional divergence between the multiple groEL gene paralogs in Chlamydiae

Background

RNA molecules, through their dual appearance as sequence and structure, represent a suitable model to study evolutionary properties of quasispecies. The essential ingredient in this model is the differentiation between genotype (molecular sequences which are affected by mutation) and phenotype (molecular structure, affected by selection). This framework allows a quantitative analysis of organizational properties of quasispecies as they adapt to different environments, such as their robustness, the effect of the degeneration of the sequence space, or the adaptation under different mutation rates and the error threshold associated.

Results

We describe and analyze the structural properties of molecular quasispecies adapting to different environments both during the transient time before adaptation takes place and in the asymptotic state, once optimization has occurred. We observe a minimum in the adaptation time at values of the mutation rate relatively far from the phenotypic error threshold. Through the definition of a consensus structure, it is shown that the quasispecies retains relevant structural information in a distributed fashion even above the error threshold. This structural robustness depends on the precise shape of the secondary structure used as target of selection. Experimental results available for natural RNA populations are in qualitative agreement with our observations.

Conclusion

Adaptation time of molecular quasispecies to a given environment is optimized at values of the mutation rate well below the phenotypic error threshold. The optimal value results from a trade-off between diversity generation and fixation of advantageous mutants. The critical value of the mutation rate is a function not only of the sequence length, but also of the specific properties of the environment, in this case the selection pressure and the shape of the secondary structure used as target phenotype. Certain functional motifs of RNA secondary structure that withstand high mutation rates (as the ubiquitous hairpin motif) might appear early in evolution and be actually frozen evolutionary accidents.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg²⁺ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg²⁺; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Branching structure in arborescent animals: Models of relative growth

Many invertebrate animals belonging to diverse phyla grow as regularly branching structures with the general appearance of miniature trees. If it is assumed that regularity of branching implies regularity in growth, models can be mathematically derived to depict growth of such a structure as a set of changing morphologic properties. Modes of growth, branching properties, and growth models can be expected to differ markedly from one major taxonomic group to another. Nevertheless, these properties can furnish a useful basis for comparing adaptive morphologies and underlying mechanical designs not only among arborescent animals, but with arborescent plants as well.Branching structures of some cheilostome bryozoans with rigidly erect, arborescent growth habits are inferred to result from continuous growth at steadily increasing numbers of growing tips through a process of repeated bifurcation and lengthening. In a model of continuous growth, the pattern by which the number of growing tips increases can be shown to be a generalized mathematical series, of which the Fibonacci series and a geometric series are two special cases. The quantities which determine the series can be calculated from measurable properties of the branching structure: lengths of paired branch portions ending in growing tips (relative growth ratio), lengths of paired branch portions between bifurcations (mean link length and link-length ratio), and numbers of branch portions belonging to different orders (branching ratio). Data for eight species of cheilostome bryozoans indicate, with high levels of confidence, that measurable branching properties and the models of relative growth inferred from them are species-specific. This specificity and a tendency to adhere to characteristic values of branching properties during growth are apparently direct expressions of internal control in these bryozoans.     

Effect of extraction parameters on the chemical structure and gel properties of kappa/iota-hybrid carrageenans obtained from Mastocarpus stellatus

Extraction parameters (temperature, pH, duration) and alkaline pre-treatment duration have been systematically varied in the aim of exploring their impact on both chemical structure and gelling properties of carrageenan biopolymers obtained from Mastocarpus stellatus seaweeds, collected on the Northern coast of Portugal. Increasing the alkaline pre-treatment duration PT leads to kappa/iota-hybrid carrageenans containing less sulphate groups and biological precursor monomers. As a result, gel properties in the presence of KCl are improved as demonstrated by the increase in the Young's modulus with parameter PT. Increasing the extraction duration t ameliorates the biopolymer yield with no significant change in the complex kappa/iota-hybrid carrageenan chemical structure. However, smaller molecular weights are obtained and gel properties are seen to be negatively affected. Extraction temperature and pH have dramatic effects on the biopolymer gel strength, and a set of extraction parameters optimized with respect to extraction yield and gel properties is reported. In addition, kappa/iota-hybrid carrageenans obtained throughout this study exhibit a wide range of gel strengths in KCl, and allow us to present correlations between gel thermal properties and the kappa/iota-hybrid carrageenans chemical structure.     

蛛网结构性能及其适应性

蛛网是蜘蛛的捕食工具,蛛网的结构性能不仅影响蜘蛛的捕食效率,也关系着蜘蛛的捕食投入。在不同的内外环境条件影响下,蜘蛛会通过蛛网结构性能上的相应变化来调整捕食策略和维持网结构的稳定性。本文主要综述了蛛网的结构性能以及蜘蛛通过蛛网结构性能表现出的对环境因子的适应性。     

Characterization of structure and properties of bone by spectral measure method

Novel mathematical method called spectral measure method (SMM) is developed for characterization of bone structure and indirect estimation of bone properties. The spectral measure method is based on an inverse homogenization technique which allows to derive information about the structure of composite material from measured effective electric or viscoelastic properties. The mechanical properties and ability to withstand fracture depend on the structural organization of bone as a hierarchical composite. Information about the bone structural parameters is contained in the spectral measure in the Stieltjes integral representation of the effective properties. The method is based on constructing the spectral measure either by calculating it directly from micro-CT images or using measurements of electric or viscoelastic properties over a frequency range. In the present paper, we generalize the Stieltjes representation to the viscoelastic case and show how bone microstructure, in particular, bone volume or porosity, can be characterized by the spectral function calculated using measurements of complex permittivity or viscoelastic modulus. For validation purposes, we numerically simulated measured data using micro-CT images of cancellous bone. Recovered values of bone porosity are in excellent agreement with true porosity estimated from the micro-CT images. We also discuss another application of this method, which allows to estimate properties difficult to measure directly. The spectral measure method based on the derived Stieltjes representation for viscoelastic composites, has a potential for non-invasive characterization of bone structure using electric or mechanical measurements. The method is applicable to sea ice, porous rock, and other composite materials.     

Adsorption of arabinoxylan on cellulosic surfaces: influence of degree of substitution and substitution pattern on adsorption characteristics

This study presents results that show that the fine structure of arabinoxylan affects its interaction with cellulosic surfaces, an important understanding when designing and evaluating properties of xylan-cellulose-based materials. Arabinoxylan samples, with well-defined structures, were prepared from a wheat flour arabinoxylan with targeted enzymatic hydrolysis. Turbidity measurements and analyses using NMR diffusometry showed that the solubility and the hydrodynamic properties of arabinoxylan are determined not only by the degree of substitution but also by the substitution pattern. On the basis of results obtained from adsorption experiments on microcrystalline cellulose particles and on cellulosic model surfaces investigated with quartz crystal microbalance with dissipation monitoring, it was also found that arabinoxylan adsorbs irreversibly on cellulosic surfaces and that the adsorption characteristics, as well as the properties of the adsorbed layer, are controlled by the fine structure of the xylan molecule.     

Structural and ligand-binding properties of a truncated form of Bacillus anthracis adenylate cyclase and of a catalytically inactive variant in which glutamine substitutes for lysine-346

A truncated, 541-residue-long, Bacillus anthracis adenylate cyclase was expressed in Escherichia coli. The purified protein (CYA 62) exhibited catalytic and CaM-binding properties identical with those of the wild-type enzyme secreted by B. anthracis. The analysis of the secondary structure of the CYA 62 protein by Fourier transform infrared spectroscopy and circular dichroism revealed the dominance of beta-type structure. The protein shows a relatively low thermal stability with the midpoint denaturation temperature at 45 degrees C. A catalytically inactive variant of CYA 62 in which Gln substituted for Lys-346 (CYA 62 K346Q) was comparatively analyzed for its secondary structure and thermal stability, as well as ligand-binding properties with fluorescent derivatives of ATP and calmodulin. The K346Q variant of CYA 62 has a similar secondary structure and comparable calmodulin binding properties to those of the parent protein and exhibits only slightly reduced thermal stability (the apparent midpoint denaturation temperature is at 43 degrees C). Despite these similarities, the binding of 3'-anthraniloyl-2'-deoxy-ATP (a fluorescent ATP analogue) to the modified protein is severely impaired, from which we conclude that the prime function of Lys-346 in the wild-type enzyme from B. anthracis is to ensure tight binding of the nucleotide substrate to the active site.     

Purification and characterization of an endo-polygalacturonase from a mutant of Saccharomyces cerevisiae.

An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.