Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Effects of morin on snake venom phospholipase A(2) (PLA(2))

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA(2) isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to. study the effect of flavonoids on PLA(2). We observed that a treatment of PLA(2) with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA(2) possess a second pharmacological site which does not affect or depend on the enzymatic activity. (c) 2005 Elsevier Ltd. All rights reserved.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM and HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34 Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and de novo amino acid sequencing, exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitre-3-(octanoyloxy)benzoic acid, determined its V(max) (7.56 nmoles/min) and K(M) (2.76 mM).Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75 mM and 0.4 mu M, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2. (c) 2008 Elsevier Ltd. All rights reserved.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Flavonoids, coumarins and other polyphenolic compounds are powerful antioxiants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alphahelical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptacline against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. on the other hand, 7-HOC exhibited a more potent inhibitory effect on sPUL2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom. (C) 2008 Published by Elsevier Ltd.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Background: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A(2) are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A(2) drugs.Methods: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.Results: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 +/- 0.28 mu g/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA(2) inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.Conclusion: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Thiolated Au18cluster: preferred Ag sites for doping, structures, and optical and chiroptical properties

Recently, the X-ray determined structure of the thiolated Au18 cluster has been reported. In this communication, we addressed a study of structures and chiroptical properties of thiolated Au18 cluster doped with up to ten Ag atoms, which have been calculated by Time Dependent Density Functional Theory (TD-DFT). The number of Ag atoms was steadily varied and more stable isomers showed optical and Circular Dichroism (CD) spectra distinct from that found for the parent Au18 cluster. Doping with more than four Ag atoms results in enhancement of the oscillator strength of the HOMO–LUMO peak and it is expected that this feature can be exploited for photoluminescence applications.

On the structure of the Au18(SR)14 cluster

First principles calculations are used for a systematic search of the lowest-energy (most-stable) structure of the recently synthesized Au18(SR)14 cluster. A comparison of the calculated optical absorption and electronic circular dichroism spectra, which are highly sensitive to the cluster structure and chirality, with the experimental spectra of the glutathione-protected gold cluster, Au18(SG)14, is used to discriminate between low-energy isomers of the Au18(SR)14 (R = CH3) cluster. From the good agreement between calculated and measured spectra, it is predicted that the structure of the Au18(SR)14 cluster consists of a prolate Au8 core covered with two dimer (SR–Au–SR–Au–SR) and two trimer (SR–Au–SR–Au–SR–Au–SR) motifs. These results provide additional evidence on the existence of longer trimer motifs as protecting units of small thiolated gold clusters.

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. (C) 2010 Elsevier B.V. All rights reserved.

The periplasmic domain of the barrel assembly machinery protein A (BamA) from Escherichia coli assists folding of outer membrane protein A

The assembly of outer membranes of the cell wall of Gram-negative bacteria and of various organelles of eukaryotic cells requires the evolutionarily conserved β-barrel-assembly machinery (BAM) complex. This thesis describes the biochemical and biophysical properties of the periplasmic domain of the β-barrel assembly machinery protein A (PD-BamA) of the E. coli BAM complex, its effect on insertion and folding of the Outer membrane protein A (OmpA) into lipid bilayers and the identification of regions of PD-BamA that may be involved in protein-protein interactions. The secondary structure of PD-BamA in mixed lipid bilayers, analyzed by Circular dichroism (CD) spectroscopy, contained less β-sheet at an increased content of phosphatidylglycerol (PG) in the lipid membrane. This result showed membrane binding, albeit only in the presence of negatively charged lipids. Fluorescence spectroscopy demonstrated that PD-BamA only binds to lipid bilayers containing the negatively charged DOPG, confirming the results of CD spectroscopy. PD-BamA did not bind to zwitterionic but overall neutral lipid bilayers. PD-BamA bound to OmpA at a stoichiometry of 1:1. PD-BamA strongly facilitated insertion and folding of OmpA into lipid membranes. Kinetics of PD-BamA mediated folding of OmpA was well described by two parallel folding processes, a fast folding process and a slow folding process, differing by 2-3 orders of magnitude in their rate constants. The folding yields of OmpA depended on the concentration of lipid membranes and also on the lipid head groups. The presence of PD-BamA resulted in increased folding yields of OmpA in negatively charged DOPG, but PD-BamA did not affect the folding kinetics of OmpA into bilayers of zwitterionic but overall neutral lipids. The efficiency of folding and insertion of OmpA into lipid bilayers strongly depended on the ratio PD-BamA/OmpA and was optimal at equimolar concentrations of PD-BamA and OmpA. To examine complexes of unfolded OmpA with PD-BamA in more detail, site-directed spectroscopy was used to explore contact regions in both, PD-BamA and OmpA. Similarly, contact regions were also investigated for another protein complex formed by PD-BamA and the lipoprotein BamD. The obtained data suggest, that the site of interaction on PD-BamA for OmpA might be oriented towards the exterior environment away from the preceding POTRA domains, but that PD-BamA is oriented with its short α-helix α1 of POTRA domain 5 towards the C-terminal end of BamD.

Chiroptical spectroscopy of biomolecules using chiral plasmonic nanostructures

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.