cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Automatic classification of enzyme family in protein annotation

Most of the tasks in genome annotation can be at least partially automated. Since this annotation is time-consuming, facilitating some parts of the process - thus freeing the specialist to carry out more valuable tasks - has been the motivation of many tools and annotation environments. In particular, annotation of protein function can benefit from knowledge about enzymatic processes. The use of sequence homology alone is not a good approach to derive this knowledge when there are only a few homologues of the sequence to be annotated. The alternative is to use motifs. This paper uses a symbolic machine learning approach to derive rules for the classification of enzymes according to the Enzyme Commission (EC). Our results show that, for the top class, the average global classification error is 3.13%. Our technique also produces a set of rules relating structural to functional information, which is important to understand the protein tridimensional structure and determine its biological function. © 2009 Springer Berlin Heidelberg.

Crystal structure of Jararacussin-I: The highly negatively charged catalytic interface contributes to macromolecular selectivity in snake venom thrombin-like enzymes

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin-like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin-I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three-dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin-I is highly negatively charged, which contributes to its unique macromolecular selectivity. © 2012 The Protein Society.

Silver colloidal nanoparticles: Effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms

Aim: The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Methods and Results: Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13·5 or 54 μg SN ml-1 for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. Conclusions: In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. Significance and Impact of the Study: This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections. © 2012 The Society for Applied Microbiology.

Gestational protein restriction induces CA3 dendritic atrophy in dorsal hippocampal neurons but does not alter learning and memory performance in adult offspring

Studies have demonstrated that nutrient deficiency during pregnancy or in early postnatal life results in structural abnormalities in the offspring hippocampus and in cognitive impairment. In an attempt to analyze whether gestational protein restriction might induce learning and memory impairments associated with structural changes in the hippocampus, we carried out a detailed morphometric analysis of the hippocampus of male adult rats together with the behavioral characterization of these animals in the Morris water maze (MWM). Our results demonstrate that gestational protein restriction leads to a decrease in total basal dendritic length and in the number of intersections of CA3 pyramidal neurons whereas the cytoarchitecture of CA1 and dentate gyrus remained unchanged. Despite presenting significant structural rearrangements, we did not observe impairments in the MWM test. Considering the clear dissociation between the behavioral profile and the hippocampus neuronal changes, the functional significance of dendritic remodeling in fetal processing remains undisclosed. © 2012 ISDN.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings. ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis. © 2013 Colombo et al.; licensee BioMed Central Ltd.

Analyzing the effect of homogeneous frustration in protein folding

The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. © 2013 Wiley Periodicals, Inc.

Diversidade genética de espécies de Xanthomonas patogênicas a citros baseada em genes avr e leucine protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Visualization of Protein Folding Funnels in Lattice Models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated from Baccharis oxyodonta on the Structure and Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus terrificus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

N-Terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions

The N-terminus of the human dihydroorotate dehydrogenase (HsDHODH) has been described as important for the enzyme attachment in the inner mitochondrial membrane and possibly to regulate enzymatic activity. In this study, we synthesized the peptide acetyl-GDERFYAEHLMPTLQGLLDPESAHRL AVRFTSLGamide, comprising the residues 33-66 of HsDHODH N-terminal conserved microdomain. Langmuir monolayers and circular dichroism (CD) were employed to investigate the interactions between the peptide and membrane model, as micelles and monolayers of the lipids phosphatidylcholine (PC), 3-phosphatidylethanolamine (PE) and cardiolipin (CL). These lipids represent the major constituents of inner mitochondrial membranes. According to CD data, the peptide adopted a random structure in water, whereas it acquired α-helical structures in the presence of micelles. The π–A isotherms and polarization- modulated infrared reflection-absorption spectroscopy on monolayers showed that the peptide interacted with all lipids, but in different ways. In DPPC monolayers, the peptide penetrated into the hydrophobic region. The strongest initial interaction occurred with DPPE, but the peptide was expelled from this monolayer at high surface pressures. In CL, the peptide could induce a partial dissolution of the monolayer, leading to shorter areas at the monolayer collapse. These results corroborate the literature, where the HsDHODH microdomain is anchored into the inner mitochondrial membrane. Moreover, the existence of distinct conformations and interactions with the different membrane lipids indicates that the access to the enzyme active site may be controlled not only by conformational changes occurring at the microdomain of the protein, but also by some lipid-protein synergetic mechanism, where the HsDHODH peptide would be able to recognize lipid domains in the membrane. - See more at: http://www.eurekaselect.com/122062/article#sthash.1ZZbc7E0.dpuf