Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β

It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.     

Bursting synchronization dynamics of pancreatic β-cells with electrical and chemical coupling

Based on bifurcation analysis, the synchronization behaviors of two identical pancreatic β-cells connected by electrical and chemical coupling are investigated, respectively. Various firing patterns are produced in coupled cells when a single cell exhibits tonic spiking or square-wave bursting individually, irrespectively of what the cells are connected by electrical or chemical coupling. On the one hand, cells can burst synchronously for both weak electrical and chemical coupling when an isolated cell exhibits tonic spiking itself. In particular, for electrically coupled cells, under the variation of the coupling strength there exist complex transition processes of synchronous firing patterns such as “fold/limit cycle” type of bursting, then anti-phase continuous spiking, followed by the “fold/torus” type of bursting, and finally in-phase tonic spiking. On the other hand, it is shown that when the individual cell exhibits square-wave bursting, suitable coupling strength can make the electrically coupled system generate “fold/Hopf” bursting via “fold/fold” hysteresis loop; whereas, the chemically coupled cells generate “fold/subHopf” bursting. Especially, chemically coupled bursters can exhibit inverse period-adding bursting sequence. Fast–slow dynamics analysis is applied to explore the generation mechanism of these bursting oscillations. The above analysis of bursting types and the transition may provide us with better insight into understanding the role of coupling in the dynamic behaviors of pancreatic β-cells.     

Identification of helix capping and β-turn motifs from NMR chemical shifts

We present an empirical method for identification of distinct structural motifs in proteins on the basis of experimentally determined backbone and ¹³C^β chemical shifts. Elements identified include the N-terminal and C-terminal helix capping motifs and five types of β-turns: I, II, I′, II′ and VIII. Using a database of proteins of known structure, the NMR chemical shifts, together with the PDB-extracted amino acid preference of the helix capping and β-turn motifs are used as input data for training an artificial neural network algorithm, which outputs the statistical probability of finding each motif at any given position in the protein. The trained neural networks, contained in the MICS (motif identification from chemical shifts) program, also provide a confidence level for each of their predictions, and values ranging from ca 0.7–0.9 for the Matthews correlation coefficient of its predictions far exceed those attainable by sequence analysis. MICS is anticipated to be useful both in the conventional NMR structure determination process and for enhancing on-going efforts to determine protein structures solely on the basis of chemical shift information, where it can aid in identifying protein database fragments suitable for use in building such structures.     

A response surface methodological study on prediction of glucosylation yields of thiamin using immobilized β-glucosidase

Response surface methodology was used to predict the glucosylation yields of thiamin using immobilized β-glucosidase. A Central Composite Rotatable Design (CCRD) of 32 experiments with immobilized β-glucosidase, thiamin, incubation period, buffer concentration and pH, as five independent variables was employed at five levels. A second-order polynomial equation was developed, the regression coefficient values of which exhibited a R² value of 0.74. Contour plots explained the glucosylation behaviour of the enzyme through a reversal in glucosylation at a cross-over point corresponding to 60% (w/w d-glucose) immobilized β-glucosidase and 0.12 mM buffer concentration at pH 6. The highest conversion yield of 58% obtained experimentally compared well with the predicted yield of 52% under optimum conditions of 40% immobilized β-glucosidase, 0.55 mmol thiamin, 96 h incubation period and 0.16 mM buffer concentration at pH 7. Validation experiments carried out at certain random predictive conditions also showed good correspondence between experimental and predictive yields.     

Contribution of surface β-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis, gtfF genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.     

A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.     

DFT study of HOMO structural map of β-diketones and β-ketoesters; towards prediction of electrochemical oxidation

In this study, anodic oxidation potentials of a training set of the selected 14 β-diketones and β-diketoesters were measured by means of cyclic voltammetry on a glassy carbon electrode and correlated with the highest occupied molecular orbital (HOMO) calculated at the #B3LYP/6-311+G** level. HOMO energy level and HOMO structural map were used to make a powerful model to classify molecules which show similar chemical and electrochemical behaviours. The linear correlation between anodic peak potential, E_pA, and E_HOMO of classified compounds, certifies that they have the same mechanism of the electron transfer reaction.     

Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.     

Application of a chemoenzymatic glycosylation method to α-chymotrypsin and Candida rugosa lipase surface modifications

A recently developed chemoenzymatic glycosylation procedure has been successfully applied on two hydrolytic enzymes, α-chymotrypsin and Candida rugosa lipase. First, a number of sucrose molecules have been bound to the surface lysine residues and then, lengthening of the glycosidic chains has been carried out by the action of a levansucrase from Bacillus subtilis. For both steps, reaction conditions have been studied in order to obtain a range of glycosylation degrees. The influence of glycoside binding on biocatalyst surface characteristics has been assessed and a progressive increase in global enzyme hydrophilic character with glycosylation has been observed. Besides, the study of hydrolytic activity and kinetic constants showed that the performed modifications brought about a certain decrease in enzyme hydrolytic activity and very slight variations in enzyme-substrate affinity.     

EU-OPENSCREEN—chemical tools for the study of plant biology and resistance mechanisms

EU-OPENSCREEN is an academic research infrastructure initiative in Europe for enabling researchers in all life sciences to take advantage of chemical biology approaches to their projects. In a collaborative effort of national networks in 16 European countries, EU-OPENSCREEN will develop novel chemical compounds with external users to address questions in, among other fields, systems and network biology (directed and selective perturbation of signalling pathways), structural biology (compound-target interactions at atomic resolution), pharmacology (early drug discovery and toxicology) and plant biology (response of wild or crop plants to environmental and agricultural substances). EU-OPENSCREEN supports all stages of a tool development project, including assay adaptation, high-throughput screening and chemical optimisation of the ‘hit’ compounds. All tool compounds and data will be made available to the scientific community. EU-OPENSCREEN integrates high-capacity screening platforms throughout Europe, which share a rationally selected compound collection comprising up to 300,000 (commercial and proprietary compounds collected from European chemists). By testing systematically this chemical collection in hundreds of assays originating from very different biological themes, the screening process generates enormous amounts of information about the biological activities of the substances and thereby steadily enriches our understanding of how and where they act.     

Hydrodynamics of Ebrié lagoon as revealed by a chemical and isotopic study

To determine the origin and the circulation of waters in the different areas of the Ebrié lagoon (Ivory Coast), ionic concentrations (K⁺ Cl^-) and isotopic (¹⁸O) measurements were performed. Sixty stations were sampled. Chemical and isotopic analyses were made thrice during a hydrological cycle: in May 1986, at the end of the great dry season; in October 1986, during the maximum of rainfall, in December 1987, after the Comoe river peak flow. From a hydrodynamical point of view, the results reported in this work indicate that the lagoon comprises four distinctive areas. The first is filled with freshwater all the year round and is characterized by an weak isotopic enrichment (these waters are of continental origin and annually renewed); the second corresponds to oligohaline waters highly enriched in¹⁸O (waters essentially of continental origin and poorly renewed); the third area is constituted of a mixture of waters of continental and oceanic origins. The latter group can be separated into two subgroups: a group completely renewed by oceanic water during the dry season and another group totally renewed by freshwater during the rainy and flood seasons.     

Cytochemical study of role of α-d-mannose- and β-d-galactose-containing glycoproteins in apoptosis

Recently, we found increased levels of -d-mannose- and -d-galactose-containing glycoproteins in plasma membrane of the apoptotic murine leukemia L1210 cells (Bilyy & Stoika 2003). That indicator was suggested to be a novel marker of apoptosis in L1210 cells. The aim of our present work was to reveal if these changes in glycoprotein expression can be common for apoptotic cells of different origin and for various ways of apoptosis induction. It was demonstrated that an elevated expression of plasma membrane glycoproteins rich in -d-mannose and -d-galactose did not depend on type of cell line and its tissue origin as well as on nature of apoptosis-inducing agent. We also found that an increase in membrane glycoprotein expression was dependent on concentration of apoptosis-inducing agent and was time-dependent. Changes in glycoproteins expression were detected as early as 9–12 hours after apoptosis induction. Two hours pretreatment of cells with non-labeled lectin decreased plasma membrane staining with corresponding peroxidase-labeled lectin, probably because of lectin-induced internalization of specific membrane glycoproteins. PSL-lectin-affinity procedure was developed for isolation of apoptotic cells from their mixed population with normal cells. Lectin-dependent agglutination analysis showed that this process occurs at much lower lectin dilutions in the apoptotic cells than in the non-apoptotic cells. Thus, we found that -d-mannose- and -d-galactose-containing glycoproteins can be used for lectinocytochemical detection, study and isolation of apoptotic cells.     

Endothelial cell surface expression of protein disulfide isomerase activates β1 and β3 integrins and facilitates dengue virus infection

Infection with dengue virus (DENV) causes diseases ranging from mild dengue fever to severe hemorrhage or shock syndrome. DENV infection of endothelial cells may cause cell apoptosis or vascular leakage and result in clinical illness of hemorrhage. However, the endothelial cell molecules involved in DENV infection and the mechanisms governing virus-cell interactions are still uncertain. Since protein disulfide isomerase (PDI) reducing function at the cell surface was shown to be required for entry of certain viruses and bacteria, we explored the role of PDI expressed on endothelial cell surface in DENV infection. Using siRNA to knock down PDI, DENV infection was reduced which could be reversed by treating cells with a reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). DENV-induced PDI surface expression was mediated through the Lys-Asp-Glu-Leu (KDEL) receptor-Src family kinase signal pathway. Furthermore, cell surface PDI colocalized with β1 and β3 integrins after DENV infection, and the activation of integrins was blocked by PDI inhibition. Finally, blockade of PDI inhibited DENV entry into endothelial cells. Our findings suggest a novel mechanism whereby surface PDI which causes integrin activation is involved in DENV entry, and DENV infection further increases PDI surface expression at later time points. These findings may have implications for anti-DENV drug design.     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

Insulin analogues with modifications in the β-turn of the B-chain

The β-turn formed by the amino acid residues 20–23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of β-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the β-turn. In two analogues, we have substituted α-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potencyca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the β-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.     

Improving the resistance of a eukaryotic β-barrel protein to thermal and chemical perturbations

β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the “mitochondrial porin family” and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for β-barrel membrane proteins, we have identified three unfavorable β-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.     

Inhibitory potential of chemical substitutions at bioinspired sites of β-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins

Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design.     

Monitoring of spine curvatures and posture during pregnancy using surface topography – case study and suggestion of method

Background

Low back and pelvic pain is one of the most frequently reported disorders in pregnancy, however etiology and pathology of this problem have not been fully determined. The relationship between back pain experienced during pregnancy and posture remains unclear. It is challenging to measure reliably postural and spinal changes at the time of pregnancy, since most imaging studies cannot be used due to the radiation burden. 3D shape measurement, or surface topography (ST), systems designed for posture evaluation could potentially fill this void. A pilot study was conducted to test the potential of monitoring the change of spine curvatures and posture during pregnancy using surface topography. A single case was studied to test the methodology and preliminarily assess the usefulness of the procedure before performing a randomized trial. The apparatus used in this study was metrologically tested and utilized earlier in scoliosis screening.

Case presentation

The subject was measured using a custom-made structured light illumination scanner with accuracy of 0.2 mm. Measurement was taken every 2 weeks, between 17th and 37th week of pregnancy, 11 measurements in total. From the measurement the thoracic kyphosis and lumbar lordosis angles, and vertical balance angle were extracted automatically. Custom-written software was used for analysis. Oswestry Low Back Pain Disability Questionnaire (ODI) was done with every measurement. The values were correctly extracted from the measurement. The results were: 50.9 ± 2.4° for kyphosis angle, 58.1 ± 2.1° for lordosis angle and 4.7 ± 1.7° for vertical balance angle. The registered change was 7.4° in kyphosis angle, 8.4° in lordosis angle and 5.5° in vertical balance angle. The calculated ODI values were between moderate disability and severe disability (22 to 58 %).

Conclusions

This case study presents that surface topography may be suitable for monitoring of spinal curvature and posture change in pregnant women. The ionizing radiation studies are contraindicated during pregnancy. Surface topography data connected with information from pain level questionnaires allows to investigate the connection between changes in posture and back pain.

     

Ankyrin recognizes both surface character and shape of the 14-15 di-repeat of β-spectrin

The spectrin-based cytoskeleton is critical for cell stability, membrane organization and membrane protein trafficking. At its core is the high-affinity complex between β-spectrin and ankyrin. Defects in either of these proteins may cause hemolytic disease, developmental disorders, neurologic disease, and cancer. Crystal structures of the minimal recognition motifs of ankyrin and β-spectrin have been determined and distinct recognition mechanisms proposed. One focused on the complementary surface charges of the minimal recognition motifs, whereas the other identified an unusual kink between β-spectrin repeats and suggested a conformation-sensitive binding surface. Using isothermal titration calorimetry and site-directed mutagenesis, we demonstrate the primacy of the inter-repeat kink as the critical determinant underlying spectrin’s ankyrin affinity. The clinical implications of this are discussed in light of recognized linker mutations and polymorphisms in the β-spectrins.