Generic Technique to Generate Large Branched DNA Complexes

The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

Biocomposites of non-crosslinked natural and synthetic polymers

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(var epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Novel tear film supplements:a potential application for synthetic protein-phospholipid complexes

Purpose: Surfactant proteins A, B, C and D complex with (phospho)lipids to produce surfactants which provide low interfacial tensions. It is likely that similar complexation occurs in the tear film and contributes to its low surface tension. Synthetic protein-phospholipid complexes, with styrene maleic anhydrides (SMAs) as the protein analogue, have been shown to have similarly low surface tensions. This study investigates the potential of modified SMAs and/or SMA-phospholipid complexes, which form under physiological conditions, to supplement natural tear film surfactants. Method: SMAs were modified to provide structural variants which can form complexes under varying conditions. Infrared spectroscopy and Nuclear Magnetic Resonance were used to confirm SMA structure. Interfacial behaviour of the SMA and SMA-phospholipid complexes was studied using Langmuir trough, du Nûoy ring and pulsating bubblemethods. Factors which affect SMA-phospholipid complex formation, such as temperature and pH, were also investigated. Results: Structural manipulation of SMAs allows control over complex formation, including under physiological conditions (e.g. partial SMAesterfication allowed complexation with dimyristoylphosphatidylcholine, at pH7). The low surface tensions of the SMAs (42mN/m for static (du Nûoy ring) and 34mN/m for dynamic (Langmuir) techniques) demonstrate their surface activity at the air-aqueous interface. SMA-phospholipid complexes provide even lower surface tensions (~2 mN/m), approaching that of lung surfactant, as measured by the pulsating bubblemethod. Conclusions: Design of the molecular architecture of SMAs allows control over their surfactant properties. These SMAs could be used as novel tear films supplements, either alone to complex with native tear film phospholipids or delivered as synthetic protein-phospholipid complexes.

Effect of the Hofmeister anions upon the swelling of a self-assembled pH-responsive hydrogel

We report the effect of a range of monovalent sodium salts on the molecular equilibrium swelling of a simple synthetic microphase separated poly(methyl methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate)-block-poly(methyl methacrylate) (PMMA88-b-PDEA223-b-PMMA88) pH-responsive hydrogel. Sodium acetate, sodium chloride, sodium bromide, sodium iodide, sodium nitrate and sodium thiocyanate were selected for study at controlled ionic strength and pH; all salts are taken from the Hofmeister series (HS). The influence of the anions on the expansion of the hydrogel was found to follow the reverse order of the classical HS. The expansion ratio of the gel measured in solutions containing the simple sodium halide salts (NaCl, NaBr, and NaI) was found to be strongly related to parameters which describe the interaction of the ion with water; surface charge density, viscosity coefficient, and entropy of hydration. A global study which also included nonspherical ions (NaAce, NaNO3 and NaSCN) showed the strongest correlation with the viscosity coefficient. Our results are interpreted in terms of the Collins model,(1) where larger ions have more mobile water in the first hydration cage immediately surrounding the gel, therefore making them more adhesive to the surface of the stationary phase of the gel and ultimately reducing the level of expansion.

Investigation and development of techniques for the characterisation of the synthetic/biological interface

The purpose of this study is to increase our knowledge of the nature of the surface properties of polymeric materials and improve our understanding of how these factors influence the deposition of proteins to form a reactive biological/synthetic interface. A number of surface analytical techniques were identified as being of potential benefit to this investigation and included in a multidisciplinary research program. Cell adhesion in culture was the primary biological sensor of surface properties, and it showed that the cell response to different materials can be modified by adhesion promoting protein layers: cell adhesion is a protein-mediated event. A range of surface rugosity can be produced on polystyrene, and the results presented here show that surface rugosity does not play a major role in determining a material's cell adhesiveness. Contact angle measurements showed that surface energy (specifically the polar fraction) is important in promoting cell spreading on surfaces. The immunogold labelling technique indicated that there were small, but noticeable differences, between the distribution of proteins on a range of surfaces. This study has shown that surface analysis techniques have different sensitivities in terms of detection limits and depth probed, and these are important in determining the usefulness of the information obtained. The techniques provide information on differing aspects of the biological/synthetic interface, and the consequence of this is that a range of techniques is needed in any full study of such a complex field as the biomaterials area.

Biological interactions with synthetic polymers

Anchorage dependent cell culture is a useful model for investigating the interface that becomes established when a synthetic polymer is placed in contact with a biological system. The primary aim of this interdisciplinary study was to systematically investigate a number of properties that were already considered to have an influence on cell behaviour and thereby establish the extent of their importance. It is envisaged that investigations such as these will not only further the understanding of the mechanisms that affect cell adhesion but may ultimately lead to the development of improved biomaterials. In this study, surface analysis of materials was carried out in parallel with culture studies using fibroblast cells. Polarity, in it's ability to undergo hydrogen bonding (eg with water and proteins), had an important affect on cell behaviour, although structural arrangement and crystallinity were not found to exert any marked influence. In addition, the extent of oxidation that had occurred during the process of manufacture of substrates was also important. The treatment of polystyrene with a selected series of acids and gas plasmas confirmed the importance of polarity, structural groups and surface charge and it was shown that this polymer was not unique among `hydrophobic' materials in it's inability to support cell adhesion. The individual water structuring groups within hydrogel polymers were also observed to have controlling effects on cell behaviour. An overall view of the biological response to both hydrogel and non-hydrogel materials highlighted the importance of surface oxidation, polarity, water structuring groups and surface charge. Initial steps were also taken to analyse foetal calf serum, which is widely used to supplement cell culture media. Using an array of analytical techniques, further experiments were carried out to observe any possible differences in the amounts of lipids and calcium that become deposited to tissue culture and bacteriological grade plastic under cell culture conditions.

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

Reciprocating power generation in a chemically driven synthetic muscle

A scalable synthetic muscle has been constructed that transducts nanoscale molecular shape changes into macroscopic motion. The working material, which deforms affinely in response to a pH stimulus, is a self-assembled block copolymer comprising nanoscopic hydrophobic domains in a weak polyacid matrix. A device has been assembled where the muscle does work on a cantilever and the force generated has been measured. When coupled to a chemical oscillator this provides a free running chemical motor that generates a peak power of 20 mW kg 1 by the serial addition of 10 nm shape changes that scales over 5 orders of magnitude. It is the nanostructured nature of the gel that gives rise to the affine deformation and results in a robust working material for the construction of scalable muscle devices.

Discovering properties of new DNA-binding activity of proteins

Protein-DNA interactions are an essential feature in the genetic activities of life, and the ability to predict and manipulate such interactions has applications in a wide range of fields. This Thesis presents the methods of modelling the properties of protein-DNA interactions. In particular, it investigates the methods of visualising and predicting the specificity of DNA-binding Cys2His2 zinc finger interaction. The Cys2His2 zinc finger proteins interact via their individual fingers to base pair subsites on the target DNA. Four key residue positions on the a- helix of the zinc fingers make non-covalent interactions with the DNA with sequence specificity. Mutating these key residues generates combinatorial possibilities that could potentially bind to any DNA segment of interest. Many attempts have been made to predict the binding interaction using structural and chemical information, but with only limited success. The most important contribution of the thesis is that the developed model allows for the binding properties of a given protein-DNA binding to be visualised in relation to other protein-DNA combinations without having to explicitly physically model the specific protein molecule and specific DNA sequence. To prove this, various databases were generated, including a synthetic database which includes all possible combinations of the DNA-binding Cys2His2 zinc finger interactions. NeuroScale, a topographic visualisation technique, is exploited to represent the geometric structures of the protein-DNA interactions by measuring dissimilarity between the data points. In order to verify the effect of visualisation on understanding the binding properties of the DNA-binding Cys2His2 zinc finger interaction, various prediction models are constructed by using both the high dimensional original data and the represented data in low dimensional feature space. Finally, novel data sets are studied through the selected visualisation models based on the experimental DNA-zinc finger protein database. The result of the NeuroScale projection shows that different dissimilarity representations give distinctive structural groupings, but clustering in biologically-interesting ways. This method can be used to forecast the physiochemical properties of the novel proteins which may be beneficial for therapeutic purposes involving genome targeting in general.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.