Structure and function of plant toxins (with emphasis on cystine knot toxins)

Plant toxins are substances produced and secreted by plants to defend themselves against predators. In a broad sense, this includes all substances that have a toxic effect on targeted organisms, whether they are microbes, other plants, insects, or higher animals. Plant toxins have a diverse range of structures, from small organic molecules through to proteins. This review gives an overview of the various classes of plant toxins but focuses on an interesting class of protein-based plant toxins containing a cystine knot motif. This structural motif confers exceptional stability on proteins containing it and is associated with a wide range of biological activities. The biological activities and structural stability offer many potential applications in the pharmaceutical and agricultural fields. One particularly exciting prospect is in the use of protein-based plant toxins as molecular scaffolds for displaying pharmaceutically important bioactivities. Future applications of plant toxins are likely to involve genetic engineering techniques and molecular pharming approaches.

Integrated study by NMR and X-ray Crystallography on the analysis of the molecular interactions in heme-binding proteins

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

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This review critically examines the development of malaria vaccine candidates and the results obtained in phase IIa and IIb efficacy evaluations since the first human trial till date. It is restricted only to proteins or protein fragments produced by peptide synthesis or DNA recombinant technology, for the simple reason that these are the only products that could be structurally evaluated. It is meant to make the scientific community aware of the shortcomings of some of these constructs with regard to their 3-dimensional structure and the need for more stringent biological/functional requirements to be met before field evaluations are initiated. Both of these factors are largely responsible for most of the failures witnessed in the past 20 years.

Factor structure of the SOCRATES questionnaire in hospitalized medical patients.

The Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES), a 19-item instrument developed to assess readiness to change alcohol use among individuals presenting for specialized alcohol treatment, has been used in various populations and settings. Its factor structure and concurrent validity has been described for specialized alcohol treatment settings and primary care. The purpose of this study was to determine the factor structure and concurrent validity of the SOCRATES among medical inpatients with unhealthy alcohol use not seeking help for specialized alcohol treatment. The subjects were 337 medical inpatients with unhealthy alcohol use, identified during their hospital stay. Most of them had alcohol dependence (76%). We performed an Alpha Factor Analysis (AFA) and Principal Component Analysis (PCA) of the 19 SOCRATES items, and forced 3 factors and 2 components, in order to replicate findings from Miller and Tonigan (Miller, W. R., & Tonigan, J. S., (1996). Assessing drinkers' motivations for change: The Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES). Psychology of Addictive Behavior, 10, 81-89.) and Maisto et al. (Maisto, S. A., Conigliaro, J., McNeil, M., Kraemer, K., O'Connor, M., & Kelley, M. E., (1999). Factor structure of the SOCRATES in a sample of primary care patients. Addictive Behavior, 24(6), 879-892.). Our analysis supported the view that the 2 component solution proposed by Maisto et al. (Maisto, S.A., Conigliaro, J., McNeil, M., Kraemer, K., O'Connor, M., & Kelley, M.E., (1999). Factor structure of the SOCRATES in a sample of primary care patients. Addictive Behavior, 24(6), 879-892.) is more appropriate for our data than the 3 factor solution proposed by Miller and Tonigan (Miller, W. R., & Tonigan, J. S., (1996). Assessing drinkers' motivations for change: The Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES). Psychology of Addictive Behavior, 10, 81-89.). The first component measured Perception of Problems and was more strongly correlated with severity of alcohol-related consequences, presence of alcohol dependence, and alcohol consumption levels (average number of drinks per day and total number of binge drinking days over the past 30 days) compared to the second component measuring Taking Action. Our findings support the view that the SOCRATES is comprised of two important readiness constructs in general medical patients identified by screening.

Modeling of electrolyte sorption – from phase equilibria to dynamic separation systems

In this thesis, general approach is devised to model electrolyte sorption from aqueous solutions on solid materials. Electrolyte sorption is often considered as unwanted phenomenon in ion exchange and its potential as an independent separation method has not been fully explored. The solid sorbents studied here are porous and non-porous organic or inorganic materials with or without specific functional groups attached on the solid matrix. Accordingly, the sorption mechanisms include physical adsorption, chemisorption on the functional groups and partition restricted by electrostatic or steric factors. The model is tested in four Cases Studies dealing with chelating adsorption of transition metal mixtures, physical adsorption of metal and metalloid complexes from chloride solutions, size exclusion of electrolytes in nano-porous materials and electrolyte exclusion of electrolyte/non-electrolyte mixtures. The model parameters are estimated using experimental data from equilibrium and batch kinetic measurements, and they are used to simulate actual single-column fixed-bed separations. Phase equilibrium between the solution and solid phases is described using thermodynamic Gibbs-Donnan model and various adsorption models depending on the properties of the sorbent. The 3-dimensional thermodynamic approach is used for volume sorption in gel-type ion exchangers and in nano-porous adsorbents, and satisfactory correlation is obtained provided that both mixing and exclusion effects are adequately taken into account. 2-Dimensional surface adsorption models are successfully applied to physical adsorption of complex species and to chelating adsorption of transition metal salts. In the latter case, comparison is also made with complex formation models. Results of the mass transport studies show that uptake rates even in a competitive high-affinity system can be described by constant diffusion coefficients, when the adsorbent structure and the phase equilibrium conditions are adequately included in the model. Furthermore, a simplified solution based on the linear driving force approximation and the shrinking-core model is developed for very non-linear adsorption systems. In each Case Study, the actual separation is carried out batch-wise in fixed-beds and the experimental data are simulated/correlated using the parameters derived from equilibrium and kinetic data. Good agreement between the calculated and experimental break-through curves is usually obtained indicating that the proposed approach is useful in systems, which at first sight are very different. For example, the important improvement in copper separation from concentrated zinc sulfate solution at elevated temperatures can be correctly predicted by the model. In some cases, however, re-adjustment of model parameters is needed due to e.g. high solution viscosity.

Dinámica del anillo mitral en pacientes con insuficiencia mitral isquemica

OBJETIVOS: Describir y comparar los cambios dinámicos de la geometría del anillo mitral durante todo el ciclo cardiaco en pacientes con insuficiencia mitral isquémica y pacientes con válvula mitral normal. MATERIALES Y MÉTODOS: Los estudios ecocardiográficos analizados fueron 37, 23 con insuficiencia mitral isquémica y 14 con válvula mitral normal. La reconstrucción del anillo se realizó en la estación de trabajo Xcelera (Philips Medial Systems) mediante la herramienta de análisis mitral (MVQ), en 5 momentos del ciclo cardiaco: Comienzo de Sístole, Mitad de Sístole, Final de Sístole, Mitad de Diástole y Final de Diástole. RESULTADOS: El anillo del grupo control, fue más dinámico, con sus menores dimensiones al final de la diástole, presentando incremento progresivo durante la sístole. Los cambios en el perímetro y el área, fueron significativos entre el comienzo y mitad de la sístole (p:0.087 y p: 0.055). En el grupo con insuficiencia mitral isquémica, el anillo fue más estático. Todas las dimensiones en este grupo, fueron mayores en los cinco momentos del ciclo cardiaco. (p < 0.1). El anillo también fue más plano, con un índice morfológico anular menor al del grupo control (p:0.087). DISCUSIÓN Y CONCLUSIONES: En pacientes sin insuficiencia mitral, el anillo es una estructura dinámica. Durante la sístole, las menores dimensiones se produjeron al comienzo de este periodo y la conformación en silla de montar se mantuvo, protegiendo contra la insuficiencia mitral. El anillo del grupo con insuficiencia mitral fue más estático y plano, perdiendo los mecanismos protectores.

Synthesis, structure and electrochemical properties of some thiosemicarbazone complexes of ruthenium

Reaction of salicylaldehyde thiosemicarbazone (L-1), 2-hydroxyacetophenone thiosemicarbazone (L-2) and 2-hydroxynapthaldehyde thiosemicarbazone (L-3) with [Ru(dmso)(4)Cl-2] affords a family of three dimeric complexes (1), (2) and (3) respectively. Crystal structure of the complex (3) has been determined. In these complexes, each monomeric unit consists of one ruthenium center and two thiosemicarbazone ligands, one of which is coordinated to ruthenium as O,N,S-donor and the other as N,S-donor forming a five-membered chelate ring. Two such monomeric units remain bridged by the sulfur atoms of the O,N,S-coordinated thiosemicarbazones. Due to this sulfur bridging, the two ruthenium centers become so close to each other, that a ruthenium-ruthenium single bond is also formed. All the complexes are diamagnetic in the solid state and in dimethylsulfoxide solution show intense absorptions in the visible and ultraviolet region. Origin of these spectral transitions has been established from DFT calculations. Cyclic voltammetry on the complexes shows two irreversible ligand oxidations on the positive side of SCE and two irreversible ligand reductions on the negative side.

Homoleptic copper(II) and copper(I) complexes of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline. Six-coordinate copper(I) in solution

Reaction of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline (L) with Cu(ClO(4))(2)center dot 6H(2)O in methanol in 3:1 M ratio at room temperature yields light green [CuL(3)](ClO(4))(2)center dot H(2)O (1). The X-ray crystal structure of the hemi acetonitrile solvate [CuL(3)](ClO(4))(2)center dot 0.5CH(3)CN has been determined which shows Jahn-Teller distortion in the CuN(6) core present in the cation [CuL(3)](2+). Complex 1 gives an axial EPR spectrum in acetonitrile-toluene glass with g(parallel to) = 2.262 (A(parallel to) = 169 x 10 (4) cm (1)) and g(perpendicular to) = 2.069. The Cu(II/I) potential in 1 in CH(2)Cl(2) at a glassy carbon electrode is 0.32 V versus NHE. This potential does not change with the addition of extra L in the medium implicating generation of a six-coordinate copper(I) species [CuL(3)](+) in solution. B3LYP/LanL2DZ calculations show that the six Cu-N bond distances in [CuL(3)](+) are 2.33, 2.25, 2.32, 2.25, 2.28 and 2.25 angstrom while the ideal Cu(I)-N bond length in a symmetric Cu(I)N(6) moiety is estimated as 2.25 angstrom. Reaction of L with Cu(CH(3)CN)(4)ClO(4) in dehydrated methanol at room temperature even in 4:1 M proportion yields [CuL(2)]ClO(4) (2). Its (1)H NMR spectrum indicates that the metal in [CuL(2)](+) is tetrahedral. The Cu(II/I) potential in 2 is found to be 0.68 V versus NHE in CH(2)Cl(2) at a glassy carbon electrode. In presence of excess L, 2 yields the cyclic voltammogram of 1. From (1)H NMR titration, the free energy of binding of L to [CuL(2)](+) to produce [CuL(3)](+) in CD(2)Cl(2) at 298 K is estimated as -11.7 (+/-0.2) kJ mol (1).

A spatio-temporal structure-based approach to drought characterisation

Drought characterisation is an intrinsically spatio-temporal problem. A limitation of previous approaches to characterisation is that they discard much of the spatio-temporal information by reducing events to a lower-order subspace. To address this, an explicit 3-dimensional (longitude, latitude, time) structure-based method is described in which drought events are defined by a spatially and temporarily coherent set of points displaying standardised precipitation below a given threshold. Geometric methods can then be used to measure similarity between individual drought structures. Groupings of these similarities provide an alternative to traditional methods for extracting recurrent space-time signals from geophysical data. The explicit consideration of structure encourages the construction of summary statistics which relate to the event geometry. Example measures considered are the event volume, centroid, and aspect ratio. The utility of a 3-dimensional approach is demonstrated by application to the analysis of European droughts (15 °W to 35°E, and 35 °N to 70°N) for the period 1901–2006. Large-scale structure is found to be abundant with 75 events identified lasting for more than 3 months and spanning at least 0.5 × 106 km2. Near-complete dissimilarity is seen between the individual drought structures, and little or no regularity is found in the time evolution of even the most spatially similar drought events. The spatial distribution of the event centroids and the time evolution of the geographic cross-sectional areas strongly suggest that large area, sustained droughts result from the combination of multiple small area (∼106 km2) short duration (∼3 months) events. The small events are not found to occur independently in space. This leads to the hypothesis that local water feedbacks play an important role in the aggregation process.

Na5(Ga4S)(GaS4)3·6H2O: a three-dimensional thiogallate containing a novel octahedral building block

The new thiogallate Na5(Ga4S)(GaS4)3·6H2O has been prepared solvothermally, using 3,5-dimethyl pyridine as a solvent, and characterised by powder and single crystal X-ray diffraction. This material, which exhibits a three-dimensional crystal structure, crystallises in the cubic space group View the MathML sourceF4¯3c (a = 17.557(4) Å). The crystal structure contains octahedral building blocks [Ga4S (GaS4)6]20−, linked into a three-dimensional network with a perovskite-type topology, and sodium hydrate clusters, [Na5(H2O)6]5+, filling the cavities in the [Ga4S(GaS4)6/2]5− framework. UV–Vis diffuse reflectance measurements indicate that this material is a wide band gap semiconductor, with a band gap of ca. 4.4 eV.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Quantitative structure-activity relationships for a series of inhibitors of cruzain from Trypanosoma cruzi: Molecular modeling, CoMFA and CoMSIA studies

Human parasitic diseases are the foremost threat to human health and welfare around the world. Trypanosomiasis is a very serious infectious disease against which the currently available drugs are limited and not effective. Therefore, there is an urgent need for new chemotherapeutic agents. One attractive drug target is the major cysteine protease from Trypanosoma cruzi, cruzain. In the present work, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) studies were conducted on a series of thiosemicarbazone and semicarbazone derivatives as inhibitors of cruzain. Molecular modeling studies were performed in order to identify the preferred binding mode of the inhibitors into the enzyme active site, and to generate structural alignments for the three-dimensional quantitative structure-activity relationship (3D QSAR) investigations. Statistically significant models were obtained (CoMFA. r(2) = 0.96 and q(2) = 0.78; CoMSIA, r(2) = 0.91 and q(2) = 0.73), indicating their predictive ability for untested compounds. The models were externally validated employing a test set, and the predicted values were in good agreement with the experimental results. The final QSAR models and the information gathered from the 3D CoMFA and CoMSIA contour maps provided important insights into the chemical and structural basis involved in the molecular recognition process of this family of cruzain inhibitors, and should be useful for the design of new structurally related analogs with improved potency. (C) 2009 Elsevier Inc. All rights reserved.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Classical and fragment-based hologram structure-activity relationships for a series of analgesic cyclic imides

Cyclic imides have been widely employed in drug design research due to their multiple pharmacological and biological properties. In the present study, two-dimensional quantitative structure-activity relationship (2D QSAR) studies were conducted on a series of potent analgesic cyclic imides using both classical and hologram QSAR (HQSAR) methods, yielding significant statistical models (classical QSAR, q(2) = 0.80; HQSAR, q(2) = 0.84). The models were then used to evaluate an external data test, and the predicted values were in good agreement with the experimental results, indicating their consistency for untested compounds.

Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.