Solution structure of Pisum sativum defensin 1 by high resolution NMR: plant defensins, identical backbone with different mechanisms of action.

Pisum sativum defensin 1 (Psd1) is a 46 amino acid residue plant defensin isolated from seeds of pea. The three-dimensional structure in solution of Psd1 was determined by two-dimensional NMR data recorded at 600 MHz. Experimental restraints were used for structure calculation using CNS and torsion-angle molecular dynamics. The 20 lowest energy structures were selected and further subjected to minimization, giving a root-mean-square deviation of 0.78(+/- 0.22) A in the backbone and 1.91(+/-0.60) A for over all atoms of the molecule. The protein has a globular fold with a triple-stranded antiparalell beta-sheet and an alpha-helix (from residue Asn17 to Leu27). Psd1 presents the so called "cysteine stabilized alpha/beta motif" and presents identical three-dimensional topology in the backbone with other defensins and neurotoxins. Comparison of the electrostatic surface potential among proteins with high three-dimensional (selected using the softwares TOP and DALI) topology gave insights into the mode of action of Psd1. The surface topologies between proteins that present antifungal activity or sodium channel inhibiting activity are different. On the other hand the surface topology presents several common features with potassium channel inhibitors, suggesting that Psd1 presents this activity. Other common features with potassium channel inhibitors were found including the presence of a lysine residue essential for inhibitory activity. The identity of Psd1 in primary sequence is not enough to infer a mechanism of action, in contrast with the strategy proposed here.     

The Genetic Algorithm Applied as a Modelling Tool to Predict the Fold of Small Proteins with Different Topologies

The genetic algorithm exploits the principles of natural evolution. Solution trials are evolved by mutation, recombination and selection until they achieve near optimal solutions [1].Our own approach has now been developed [2] after a general overview on the application potential for protein structure analysis [3] to a tool to delineate the three-dimensional topology for the mainchain of small proteins [4], no matter whether they are largely helical, are mixed or -strand rich [5].Results on several protein examples for these different modelling tasks are presented and compared with the experimentally observed structures (RMSDs are around 4.5-5.5 Å). To start a modelling trial only the protein sequence and knowledge of its secondary structure is required. The fittest folds obtained after the evolution at the end of the simulations yield the three dimensional models of the fold. Current limitations are protein size (generally less than 100 aminoacids), number of secondary structure elements [7-8] and irregular topologies (e.g. ferridoxins).Further, preliminary results from current simulations are illustrated. We now want to apply simple experimental or other information, which is available long before the three-dimensional structure of the protein becomes known, to refine the modelling of the protein fold and tackle also more difficult modelling examples by our tool.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020304     

Structure validation by Calpha geometry: phi,psi and Cbeta deviation

Geometrical validation around the Calpha is described, with a new Cbeta measure and updated Ramachandran plot. Deviation of the observed Cbeta atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cbeta deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new phi,psi plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the gamma-turn conformation near +75 degrees,-60 degrees, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the gamma-turn H-bond. Favored and allowed phi,psi regions are also defined for Pro, pre-Pro, and Gly (important because Gly phi,psi angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of alpha-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage).     

Experimental and theoretical DFT (B3LYP,X3LYP,CAM-B3LYP and M06-2X) study on electronic structure,spectral features,hydrogen bonding and solvent effects of 4-methylthiadiazole-5-carboxylic acid

Detailed structural, electronic and spectroscopic study of 4-methylthiadiazole-5-carboxylic acid, one of the simplest 1,2,3-thiadiazole derivatives has been performed using density functional theory at four different functionals (B3LYP, X3LYP, CAM-B3LYP and M06-2X). The two possible conformers and their dimeric forms have been investigated for the stability and hence for the calculation of molecular properties of the title compound. Vibrational analysis has been performed with the help of experimental FT-IR and FT-Raman spectra. NBO analysis has been performed to estimate the N–H—O=C hydrogen bond strength and to evaluate the intra and inter molecular charge transfer in the system. Intermolecular hydrogen-bond strength has also been computed using Atoms in Molecules (AIM) theory. To visualise spatial domain, key sites of electron transitions and electron density difference between ground as well as excited states, and their 2D and 3D plots have been computed. Solvent effect on the intermolecular hydrogen bonding have also been investigated using solvents of different polarities. Non-linear optical properties, molecular electrostatic potential surface map (MESP), thermodynamic potentials at different temperatures have also been computed and plotted.     

淡紫拟青霉胞外多糖的分离、纯化及结构分析

淡紫拟青霉NH-PL-03菌株的胞外多糖粗提物对枯萎病病原菌-尖孢镰刀菌具有较好的抑制效果,文中对淡紫拟青霉胞外多糖进行了分离纯化和结构分析,以期为其构效关系研究奠定基础。采用乙醇沉淀法从淡紫拟青霉发酵液中提取粗多糖,经Sevage法脱蛋白后,过Superdex-G75凝胶层析柱分离得到胞外多糖EP-1。紫外分光法和Sephacryl S-200 HR凝胶层析柱检测EP-1为均一多糖,Sephacryl S-200柱层析测得EP-1的分子量为35.2 kDa,完全酸水解后纸层析检测EP-1的单糖组成中仅有葡萄糖,红外光谱、高碘酸氧化和Smith降解结果表明EP-1的化学结构是以β-(1,3)糖苷键连接而成的无分枝的葡聚糖。刚果红络合试验表明EP-1在稀的碱溶液中以3股螺旋构象存在。     

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.     

Amplification,sequencing and characterization of pectin methyl esterase inhibitor 51 gene in Tectona grandis L.f.

Tectona grandis L.f. (Teak), a very important source of incomparable timber, withstands a wide range of tropical deciduous conditions. We achieved partial amplification of pectin methylesterase inhibitor 51 (PMEI) gene in teak by E. pilularis cinnamoyl Co-A reductase (CCR) gene specific primer. The amplified teak gene was of 750 bp, 79% identity and 97% query cover with PMEI of Sesamum indicum. The phylogenetic tree clustered the amplified gene with PMEI of database plant species, Erythranthe guttata and Sesamum indicum (87% bootstrap value). On conversion to amino acid sequence, the obtained protein comprised 237 amino acids. However, PMEI region spanned from 24 to 171 amino acids, 15.94 kDa molecular weight, 8.97 pI value and C₆₉₇H₁₁₁₇N₁₉₉O₂₁₁S₉ molecular formula with four conserved cysteine residues as disulfide bridges. 25.9 % protein residues were hydrophilic, 42.7% hydrophobic and 31.2% neutral. Teak 3D PMEI protein structure corresponded well with Arabidopsis thaliana and Actinidia deliciosa PMEIs. The gene maintains integrity of pectin component of middle lamella of primary cell wall and confers tolerance against various kinds of stresses. Teak conferred with overexpression of PMEI may secure a wide adaptability as well as luxuriant timber productivity and quality in adverse/ fluctuating/ scarce climatic and environmental conditions of tropical forests.     

RNA2D3D: A program for Generating,Viewing, and Comparing 3-Dimensional Models of RNA

Abstract

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

A strategy for determining the orientations of refractory particles for reconstruction from cryo-electron micrographs with particular reference to round, smooth-surfaced, icosahedral viruses

Cryo-electron microscopy and three-dimensional image reconstruction are powerful tools for analyzing icosahedral virus capsids at resolutions that now extend below 1 nm. However, the validity of such density maps depends critically on correct identification of the viewing geometry of each particle in the data set. In some cases-for example, round capsids with low surface relief-it is difficult to identify orientations by conventional application of the two most widely used approaches-"common lines" and model-based iterative refinement. We describe here a strategy for determining the orientations of such refractory specimens. The key step is to determine reliable orientations for a base set of particles. For each particle, a list of candidate orientations is generated by common lines: correct orientations are then identified by computing a single-particle reconstruction for each candidate and then systematically matching their reprojections with the original images by visual criteria and cross-correlation analysis. This base set yields a first-generation reconstruction that is fed into the model-based procedure. This strategy has led to the structural determination of two viruses that, in our hands, resisted solution by other means.     

Identification,classification, and analysis of beta-bulges in proteins.

A beta-bulge is a region of irregularity in a beta-sheet involving two beta-strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in beta-sheets were identified and classified automatically, extending the definition of beta-bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574-2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of beta-bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, beta-bulges produce two main changes in the structure of a beta-sheet: (1) disrupt the normal alternation of side-chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands.     

Influence of Environmental Heterogeneity on the Structure of Testate Amoebae (Protozoa,Rhizopoda) Assemblages in the Plankton of the Upper Paraná River Floodplain,Brazil

In freshwater environments, testate amoebae are usually found associated with macrophytes and sediments and many studies have suggested that their occurrence in plankton samples is accidental. This implies that predictable patterns detected in planktonic assemblages should not be observed in testate amoebae assemblages. This hypothesis was tested in this study. Plankton samples were collected from different environments in the Upper Paraná River floodplain. Results show that patterns in species composition and abundance of testate amoebae are predictable, and that dominant species tend to present characteristic shell morphology in hydrologically different environments. We suggest that testate amoebae must be routinely included in plankton ecology studies, at least in floodplain environments.     

Developments,applications, and prospects of cryo‐electron microscopy

Cryo‐electron microscopy (cryo‐EM) is a structural biological method that is used to determine the 3D structures of biomacromolecules. After years of development, cryo‐EM has made great achievements, which has led to a revolution in structural biology. In this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐EM are systematically reviewed. In addition, the new development direction of cryo‐EM—cryo‐electron tomography (cryo‐ET), is discussed in detail. Also, cryo‐EM is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐EM and drug development. This review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐EM, and to bringing them new insights.     

Detection and identification of cancerous murine fibroblasts,transformed by murine sarcoma virus in culture,using Raman spectroscopy and advanced statistical methods

Background

Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer.

Methods

Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer.In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells.

Results

Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80–85% success rate based on their Raman shift spectra.

Conclusions

The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites.

General significance

Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.     

Structural,thermodynamic, and phosphatidylinositol 3‐phosphate binding properties of Phafin2

Phafin2 is a phosphatidylinositol 3‐phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ～28 kDa with an intensity‐average hydrodynamic diameter of ～7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ～40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding‐unfolding curves, obtained using urea‐ and guanidine hydrochloride‐mediated denaturation, indicate that Phafin2 undergoes a two‐state native‐to‐denatured transition. Analysis of these transitions shows that the free energy change for urea‐ and guanidine hydrochloride‐induced Phafin2 denaturation in water is ～4 kcal mol^?1. PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P‐binding domains of the protein in endomembrane compartments.     

Discovering structural correlations in alpha-helices.

We have developed a new representation for structural and functional motifs in protein sequences based on correlations between pairs of amino acids and applied it to alpha-helical and beta-sheet sequences. Existing probabilistic methods for representing and analyzing protein sequences have traditionally assumed conditional independence of evidence. In other words, amino acids are assumed to have no effect on each other. However, analyses of protein structures have repeatedly demonstrated the importance of interactions between amino acids in conferring both structure and function. Using Bayesian networks, we are able to model the relationships between amino acids at distinct positions in a protein sequence in addition to the amino acid distributions at each position. We have also developed an automated program for discovering sequence correlations using standard statistical tests and validation techniques. In this paper, we test this program on sequences from secondary structure motifs, namely alpha-helices and beta-sheets. In each case, the correlations our program discovers correspond well with known physical and chemical interactions between amino acids in structures. Furthermore, we show that, using different chemical alphabets for the amino acids, we discover structural relationships based on the same chemical principle used in constructing the alphabet. This new representation of 3-dimensional features in protein motifs, such as those arising from structural or functional constraints on the sequence, can be used to improve sequence analysis tools including pattern analysis and database search.