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Malm, Linus. "Size determination of hyaluronan and multivariate analysis of amyloid prone proteins." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-46601.
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Background.The extracellular matrix surrounds all cells within our bodies. The glycosaminoglycan hyaluronan is a major component in the extracellular matrix. Despite its structural simplicity it has been shown to be involved in several important functions. It is a lubricant and shock absorber, as well as an important player in inflammation and tumor invasion. Many of its functions are closely related to its size and concentration in tissues. Therefore methods for measuring these properties are of great importance to properly understand the role that hyaluronan play in different events. Proteins are found both inside and outside cells, and they have a wide variety of functions. The protein structure and function is determined by the properties of their building blocks, the amino acids. Several diseases have been linked to changes in the amino acid sequence of certain proteins by mutations, causing the proteins to form extracellular deposits of structures called amyloid aggregates. The aim of this thesis is to investigate the function of hyaluronan in cell cultures, develop new methods for size determination hyaluronan and to use multivariate methods to provide prediction and better understanding of factors driving protein amyloid aggregation. Methods.Cardiomyocytes and fibroblast were cultured and stimulated by different growth factors. Hyaluronan was purified and its size and concentration were measured. Crosstalk between cardiomyocytes and fibroblast were investigated and gene expression of hyaluronan synthases was determined. A new method for size measurement of hyaluronan was developed. The amyloid aggregation rate of different mutants of acylphosphatase was predicted by multivariate analysis. Results. Cardiomyocytes stimulated by PDGF-BB produced hyaluronan. Cardiomyocytes could induce fibroblast to increase its hyaluronan production, through an unknown soluble factor. The cardiomyocyte gene expression changed when stimulated by hyaluronan. GEMMA was presented as a new method for size determination of hyaluronan. Amyloid aggregation of different acylphosphatase mutants could be predicted using a multivariate regression model of the physicochemical and structural properties of the amino acid sequence. Conclusion. It was shown that cardiomyocytes are not only able to produce hyaluronan, but also induce an increased hyaluronan production in other cells. GEMMA was proven suitable for size determination of hyaluronan at very low concentrations. Multivariate analysis showed that hydrophobic patterns and charge where the most important factors for amyloid aggregation of acylphosphatase.
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Berger, Z. "The biology of aggregate-prone proteins and possible therapeutic interventions against their toxicity." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596587.
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The main aim of my project was to investigate pathways involved in protecting against the toxicity caused by different aggregate-prone proteins and to explore possible therapeutic interventions. My strategy was to generate a Drosophila model expressing a stretch of long polyalanines. This model can be used along with models of polyglutamine diseases to study various pathways in the context of different aggregate-prone proteins. In, addition, generation of this model will also help to better understand the toxicity of long polyalanines. My results show that expanded polyalanines are toxic in vivo in Drosophila and this is accompanied by aggregate formation. This suggests that polyalanines can cause diseases by a gain-of-function mechanism in vivo and that this mechanism should be considered for all diseases caused by expanded polyalanines. In order to investigate if manipulation of the Wnt and TOR pathway would be beneficial in the context of different aggregate-prone proteins in vivo, I used fly models of these disorders. Inhibition of GSK3 using lithium and a GSK3-specific inhibitor decreased toxicity of both proteins and similar effects were seen when downstream targets were manipulated in vivo. Inhibition of TOR by rapamycin decreased toxicity of proteins with long polyglutamines and polyalanines in vivo and this in vivo effect can be largely attributed to the induction of autophagy. Rapamycin also reduces toxicity of wild-type and mutant tau in vivo and these effects can be accounted for by reductions in insoluble tau. Thus, lithium, GSK3-specific inhibitors and rapamycin may be a beneficial therapeutic strategy in diseases associated with different aggregate-prone proteins.
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Gallo, Annastassia Dawn. "Homeostasis and trafficking of hydrolysis-prone metals in cells, proteins, and small molecules." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/568230.
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ChemistryPh.D.
Nature uses inorganic elements for biological processes based on the useful chemistry, abundance, and availability of each metal. Transition metals are critical in the biogeochemical cycling of essential elements and the bioinorganic chemistry of organisms. Hydrolysis-prone metals such as iron and titanium are abundant on Earth but are mostly insoluble in oxic aqueous environments. Nearly every organism requires iron for survival, therefore Nature evolved to stabilize iron from hydrolysis and hydrolytic precipitation through protein and small molecule mechanisms. Like iron, titanium primarily exists as insoluble mineral oxides and is second only to iron as the most abundant transition metal in the Earth’s crust. Despite the reputation as an inert and insoluble metal, titanium can be solubilized and made bioavailable through by chemical and biological weathering. Currently there is no known native role for titanium, however it is quite bioactive. As a stronger Lewis acid, titanium can compete with iron in binding to biomolecules and proteins. It is of interest to investigate the interactions between hydrolysis-prone metals and biological systems, from whole cell organisms to proteins and small molecules. The non-pathogenic bacterium Rhodococcus ruber GIN-1 was isolated for its ability to strongly adhere to titanium dioxide (TiO2) over other metal oxides, providing an opportunity to study the interactions between whole bacterial cells and metal oxides. The GIN-1 strain incorporates Ti(IV) ions into its biomass after adherence to anatase, rutile, and a mixture of the two morphologies. Six metals were quantitated in TiO2-exposed and control (unexposed) cells by inductively coupled plasma optical emission spectroscopy. The exposure to TiO2 caused a significant uptake of titanium with concomitant loss of iron, zinc, and possibly manganese. A collaborative project with the Strongin laboratory at Temple University works to develop stable, biomaterial photocatalysts for environment remediation of toxic inorganic contaminants. Ferritins are a class of proteins that mineralizes and stores iron as a non- toxic ferrihydrite nanoparticle. These proteins can be photoactivated with ultraviolet light to release iron from its core to remediate environmental contaminants. Ferritin can be sensitized with plasmonic gold nanoparticles to extend the photoactivity of the catalyst to the visible spectrum. Work in this thesis highlights the contribution to this collaboration from the Valentine laboratory, included the expression and purification of proteins in E. coli (human H-chain ferritin, human L-chain ferritin, and bacterial DNA protection from starved cells protein), mutation of proteins to improve sensitization of catalyst, and biomineralization with iron and titanium. The trafficking of hydrolysis prone metals is vital for the survival of nearly every organism. Iron transport proteins such as transferrins are studied to understand how nature utilizes a difficult essential metal across the domains of life. Most transferrins have two homologous lobes and are believed to have evolved from a gene duplication of a monolobal transferrin. The ascidian Ciona intestinalis has genes for both a bilobal and monolobal transferrin. Nicatransferrin (nicaTf), the monolobal transferrin from C. intestinalis, is a primitive protein that may provide insight on the evolution of transferrins in higher organisms. It is advantageous to use E. coli expression systems to produce recombinant proteins, however protein misfolding and aggregation can be a concern. To improve expression of nicaTf in E. coli, codon optimization and disulfide bonded protein expression were used. Finally, siderophores are small, high affinity iron-chelating molecules secreted from lower organisms that scavenge iron in iron-limiting conditions. R. ruber GIN-1 and R. ruber DSM 43338 strains both secrete siderophores in artificial seawater media. There are several siderophores identified from Rhodococcus species, however none have been reported from any R. ruber strain. A new siderophore was isolated and preliminary work has been done to purify and characterize the molecule. Understanding the siderophore- metal ion interactions may help elucidate the mechanism of how R. ruber cells obtain titanium from the metal-oxide particles.
Temple University--Theses
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Williams, Andrea. "Characterisation of novel autophagy pathways : implications in the clearance of disease-causing aggregate-prone proteins." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612132.
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Vincenz-Donnelly, Lisa Verfasser], and Franz-Ulrich [Akademischer Betreuer] [Hartl. "How the mammalian endoplasmic reticulum handles aggregation-prone β-sheet proteins / Lisa Vincenz-Donnelly ; Betreuer: Franz-Ulrich Hartl." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1122019467/34.
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Born, Ariane. "Etablierung und Optimierung der Error-Prone-PCR und eines Aktivitätsscreenings für Styrol-Monooxygenasen." Master's thesis, Technische Universitaet Bergakademie Freiberg Universitaetsbibliothek "Georgius Agricola", 2011. http://nbn-resolving.de/urn:nbn:de:bsz:105-qucosa-77143.
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Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurdeStyrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density
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Liu, Xueyun, and Xueyun Liu. "Probe Ca2+/Camodulin reguation of membrane proteins engineering." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/chemistry_theses/59.
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Calmodulin (CaM) is a eukaryotic Ca2+ signaling protein which can interact with more than 300 enzymes in the cell including membrane proteins Ryanodine receptor1 (RyR1) and gap junction protein connexin 43 (Cx43). By binding to Ca2+, CaM undergoes a conformational change which exposed the hydrophobic patch that access to the target protein. wt-CaM and three mutant CaM (isolate C and N domain of CaM, deletion of five residues from the central linker of CaM) are designed for studying the specific contributions to calcium binding affinity and calcium induced conformational change. wt-CaM exhibits metal binding affinity to calcium analog Tb3+ with a Kd of 3.97 nM using FRET assay and metal-buffer system and activates target protein phosphodiesterase assay. The Kd values of domain specific calcium binding affinity of CaM probed by intrinsic Phe or Tyr are 12.2 and 2.77 uM, respectively. In addition, Ca2+ also induces helicity for both w.t. CaM and C-terminal domain variant. Further, conditions such as medium and Glucose amount for isotopic labeling of CaM by 15N, 13C and D2O have been optimized with relatively high yield of hetero-isotopic labeled CaM. This prepared us to probe the detailed interaction of CaM and its target protein and calcium induced conformational change by high resolution NMR. Furthermore, RyR1 mini domain, which contains two CaM binding regions of RyR1 was designed to study the binding mode of the two regions with CaM. Obtaining bacterial expressed and purified RyR1 mini domain was achieved by engineered with a His-Tag which overcomes the insoluble issue that occurred in the initial study of expression and purification with a GST tag. Moreover, to probe the interaction of CaM to the cytosolic loop of Cx43 that contains two putative CaM binding sites as well as the role of transmembrane region of Cx43, we have successfully expressed and purified fragments Cx4388-154 and Cx4399-154 as a His-tag protein encompassing regions 88-154 and 99-154 of Cx43 with predicted CaM binding sites with and without additional transmembrane region from Cx43. Both fragments were obtained with high yield after expressed as inclusion body and His-tag purification. Fragment Cx4388-154 was shown to bind dansylated CaM with a Kd of 0.107 μM using florescence spectroscopy.
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Mossuto, Maria Francesca. "Protein amyloidogenesis: characterization of aggregation prone conformations and fibrils structure." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425566.
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Current interest in studying amyloid fibrils arises from their involvement in different fields (Chiti and Dobson, 2006). First, they play a crucial role in disorders such as Alzheimer's and Parkinson's diseases. Second, since it has been demonstrated that all polypeptide chains form fibrils under appropriate conditions, the understanding of why and how this process happens has become central problem in protein knowledge. Last, the ordered ultrastructure characterizing amyloid fibrils may be thought as a basis for nanomaterials with possible technological applications. However, despite the ability of most proteins to form amyloid fibrils, very little is known about their structures and the factors that govern their formation.
The process of amyloid formation requires the partial unfolding of protein molecules into such conformations able to interact to each others and reorganize into well-ordered structured aggregates, named amyloid fibrils.
In this Thesis the amyloid formation by globular proteins has been analyzed from different points of view, focusing in the first part of the research work on the elucidation of some conformational features promoting protein aggregation. The second part was concentrated on the characterization of the final supramolecular structure of amyloid fibrils.
In order to study the partially folded state, two globular proteins have been analyzed, alpha-lactalbumin (LA) and HypF-N. Indeed, under specific conditions, these proteins populate a not fully folded state previously shown to play an important role in the amyloid formation (Uversky, 2002; Chiti et al., 2001).
The study conducted on LA has been based on the effects of the proteolytic dissection of the molecule on its conformational features and aggregation properties. It was previously shown that LA is able to form fibrils morphologically indistinct from the pathological ones (Uversky et al., 2002). Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the ?-subdomain of the native protein (1-40/53-123, named des?-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. The main conclusion of this work was that the inherent flexibility of the LA derivatives allows the large conformational changes required to form the cross-?-structure of the amyloid fibrils. It has been emphasized that proteolysis can be considered a causative mechanism of protein aggregation and fibrillogenesis (Polverino de Laureto et al., 2005).
In the other case, the conformational characterization of an amyloidogenic state of HypF-N has been performed at acid pH, in order to allow the protein to populate a partially unfolded ensemble. Combining different biophysical and biochemical techniques, it has been shown that this partially unfolded structure has all the hallmarks of a pre-molten globule state, i.e. it is more compact than a random coil-like state but less organized than a native-like intermediate or a MG state (Uversky, 2002). Furthermore, it is shown that a modulation of the total ionic strength of the solution allows enhancing the apparent rate of aggregation of HypF-N under these conditions. This increased rate of aggregation has been shown to be mediated by the interaction of monomers to form initial oligomers, through a particular region in the sequence, corresponding to the sequence part having highly hydrophobicity, the highest beta-sheet propensity and with no net charge at acid pH, representing the ideal segment suitable to mediate protein oligomerization.
From all these studies, it is clear that, except the unique native state of globular proteins wherein the side chains pack together in a unique manner, every state of a polypeptide molecule is a broad ensemble of often diverse conformations. It is not surprising, therefore, that even the fibrillar products of aggregation processes are characterized by morphological and structural diversity, representing variations on a common theme.
The second part of my PhD Thesis deals with the structural characterization of fibrils. Many studies have been conducted on amyloid aggregates formed under different conditions by peptides, such as A?, TTR and prion fragments (Kodali and Wetzel, 2007). Indeed, the problem of amyloid formation by a full-length protein is more complex, since the dense packing reachable in amyloid fibrils made of peptides (10-40 residues) could not be accomplished in all the amino acid residues of a full-length protein, except in the core regions (Chatani and Goto, 2005). The object of my study was human lysozyme due, most of all, to the fact that some natural variants of human lysozyme (HuL) are responsible for the formation of amyloid plaques in vivo, in a so called familial non-neuropathic systemic amyloidosis (Pepys et al., 1993; Booth et al., 1997). Moreover, it has been possible to exploit the available wealth of structural and folding information about wild type HuL, since it has been shown to be able to form fibrils quite similar to the pathological ones, under acidic conditions and high temperature (Morozova-Roche et al, 2000). With respect to fibrils made of peptides, besides, studying amyloid fibrils conformations from HuL is more challenging because it is a 130 amino acid chain with the structural constrains given by the four disulfide bridges present in the lysozyme molecule. This study can also give some insights into the complex problem of strains diversity, such as for prion diseases, helping the clarification of the structural principles of amyloid fibrils which can produce multiple and distinct amyloid conformations from one protein sequence.
In the presented study, fibrils of wild-type HuL formed at low pH have been analyzed by limited proteolysis experiments and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates (Frare et al., 2006). After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive ?-sheet structure and substantial elements of non ?-sheet or random structure that are reduced significantly in the fibrils after digestion. The sequence 32-108 includes the ?-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme (Frare et al., 2004). The present data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain.
HuL variants, however, aggregate in a physiological environment, roughly at pH 7-7.5 at 37 °C, because of their instability (Dumoulin et al., 2005). In my work, it has been demonstrated that also HuL is able to aggregate under conditions similar to the pathological ones, presumably neutral pH and 37 °C. Considering that HuL forms amyloid fibrils in such different conditions (pH 2.0 50°C and pH 7.5, 60°C), a comparison of the structure and the stability of fibrils obtained under these different conditions has been conducted. In this study HuL fibrils were produced at acidic and at neutral pH, leading both to the formation of fibrils having the three hallmarks of amyloid, that are cross-beta structure, binding of ThT and an overall amyloid fiber morphology. These fibrils have been studied by means of ANS binding, FTIR and X-ray fiber diffraction in order to characterize the differences in the structure. Guanidinium-induced fibrils dissociation, instead, has been applied in order to test the chemical stability of the two kinds of fibrils. The results clearly indicate that the solution conditions used for lysozyme aggregation promote the formation of fibrils with different structural features and stability properties, due to the diverse rearrangements of the lysozyme polypeptide chain into the fibril structure.
In conclusion, the research work conducted in this Thesis allowed the comprehension of important aspects of the unfolding of some globular proteins leading to amyloid fibrils. In addition, original data have been obtained on the structural polymorphism of amyloid fibrils.
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SOUZA, Renata Silva Cabral de. "Avalia??o do potencial antioxidante e antimicrobiano de prote?nas do soro de leite concentradas por membranas e hidrolisadas por diferentes enzimas comerciais." Universidade Federal Rural do Rio de Janeiro, 2013. https://tede.ufrrj.br/jspui/handle/jspui/2534.
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The aim of this study was to evaluate the process of protein concentration in bovine whey proteins by ultrafiltration process and subsequently the protein hydrolysate obtained by enzymatic hydrolysis to produce bioactive peptides with potential antimicrobial and antioxidant activities. For concentration process was used a ceramic ultrafiltration membrane with a molecular range cut-off of 10-20 kDa, transmembrane pressure of 5 bar and, temperature of 30 ?C, 40 ?C and 50 ?C . The optimum temperature condition was at 40 ?C. The Volume Concentrate Factor (VCF) parameter was used as a end-point of the ultrafiltration process and fixed at 2, corresponding on concentrating the initial volume twice, in volume. At the temperature of 40 ?C, VCF had a correspondence on final protein concentration on the concentrated fraction by ultrafiltration and confirmed by Bradford method. Two commercial enzymes were tested Alcalase, Flavourzyme and an equivalent mixture of both 50:50 (w/w) in the hydrolysis reaction. The hydrolysis conditions were determined according to manufacturer instructions and confirmed by other studies: 60 ?C and pH 8 for Alcalase; 50 ?C and pH 7 for Flavourzyme; 50 ?C and pH 8 for enzyme mixture with enzyme / substrate ratio (w / w) 5/100 for all enzymes. The reaction was monitored by pH Stat method. The final Degree of Hydrolysis (DH) achieved was 15%, 52% and 63% for Flavourzyme, Alcalase and enzyme mixture, respectively. Five aliquots were collected along the hydrolysis for each enzyme reaction corresponding to differents DH in order to evaluatethe antioxidant activity by ORAC and ABTS assays with final values between 597- 1092 m? TE (ABTS) and from 1615 to 2920 ?M TE (ORAC) for Flavourzyme; 998-6290 ?M TE (ABTS) and 3092-7567 ?M TE ( ORAC) for Alcalase and finally 913-2678 ?M TE (ABTS) and 2547-5903 ?M TE (ORAC) for the enzyme mixture. The samples from all hydrolysates showed no antimicrobial activity against strains of Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) and Listeria monocytogenes (ATCC 9117).
A proposta do presente trabalho foi avaliar a concentra??o das prote?nas do soro de leite bovino por ultrafiltra??o e posterior obten??o de hidrolisados proteicos deste concentrado via hidr?lise enzim?tica visando obter pept?deos bioativos com potencial atividade antimicrobiana e antioxidante. Para concentra??o das prote?nas do soro foi utilizada membrana cer?mica de ultrafiltra??o com massa molar de corte de 10-20 kDa, press?o aplicada ? membrana de 5 bar, temperaturas testadas (30 ?C, 40 ?C e 50 ?C) . A temperatura ?tima selecionada foi de 40 ?C. O Fator de Concentra??o Volum?trica foi o par?metro utilizado para indicar o final do processo de ultrafiltra??o sendo fixado em duas vezes o volume inicial da alimenta??o. Na temperatura de 40 ?C foi obtida correspond?ncia entre a concentra??o volum?trica e a concentra??o proteica final na fra??o retida pela UF, que tamb?m foi o dobro da encontrada na fra??o alimenta??o, avaliada pelo m?todo de Bradford. Foram testadas duas enzimas comerciais: Alcalase, Flavourzyme e uma mistura equivalente de ambas, na propor??o 50:50 (m/m) na rea??o de hidr?lise. As condi??es de rea??o enzim?tica foram determinadas de acordo com instru??es do fabricante e corroboradas por outros estudos em: 60 ?C, pH 8 para Alcalase; 50 ?C, pH 7 para Flavourzyme; 50 ?C, pH 8 para mistura enzim?tica e rela??o enzima/substrato (g/g) foi de 5/100 para todas as enzimas. A rea??o de hidr?lise foi monitorada pelo m?todo pH Stat. Os Graus de Hidr?lise (GH) finais alcan?ados foram de 15 %, 52 % e 63 % para Flavourzyme, mistura enzim?tica e Alcalase, respectivamente. Foram coletadas cinco al?quotas correspondentes a diferentes GH ao longo da rea??o para cada condi??o enzim?tica utilizada e avaliadas quanto a atividade antioxidante pelos m?todos ABTS e ORAC tendo valores entre 597 a 1092 ?M TE (ABTS) e 1615 a 2920 ?M TE (ORAC) para Flavourzyme, 998 a 6290 ?M TE (ABTS) e 3092 a 7567 ?M TE (ORAC) para Alcalase e por fim, 913 a 2678 ?M TE (ABTS) e 2547 a 5903 ?M TE (ORAC) para a mistura enzim?tica. Nenhuma das amostras de hidrolisado com diferentes GH apresentou atividade antimicrobiana contra cepas de Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) e Listeria monocytogenes (ATCC 9117).
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Wikström, Malin. "Synthesis and protein curing abilities of membrane glycolipids." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1361.
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There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.
Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.
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Robertson, Neil. "Development and application of simple FRET-based methods for aggregation-prone LIM domain interactions." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/16912.
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LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins are important mediators of cell specification, proliferation and differentiation. These transcription factors all contain two tandem LIM domains (LIM1+2), which are non-classical zinc finger motifs that mediate protein-protein interactions. Many co-factors of these proteins contain LIM interacting domains (LIDs). The LID is a ~30-residue intrinsically disordered region (IDR) that folds upon binding to LIM1+2 domains. LID:LIM1+2 interactions and the competition established through different combinations of different binding partners play an important role in neural development and breast cancer. The ability to estimate affinities for these interactions would help provide mechanistic insight into LMO and LIM-HD complex formation and regulation. However, the propensity of LIM1+2 domains from LMO/LIM-HD proteins to aggregate and precipitate during recombinant protein production have made it difficult to measure binding affinities for LID:LIM1+2 interactions. This thesis outlines the design, optimisation and application of a series of Förster Resonance Energy Transfer (FRET)-based approaches to study LID:LIM1+2 interactions. LIM1+2 aggregation is prevented by tethering the domains to a LID using a flexible polypeptide linker. The interacting domains are in turn fused to fluorescent proteins that are optimised for FRET. Specific proteolytic cleavage of the linker allows equilibrium binding constants and dissociation rates to be determined using homologous competition and dilution-based approaches. Through the application of these simple FRET-based binding methods, this thesis reveals previously unappreciated and unknown properties of LMO and LIM-HD proteins. This work provides tools for studying other aggregation-prone proteins, as well as general implications for the activity of transcription factors and IDR interactions.
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Taylor, Christopher George. "Novel fluorescence techniques to probe protein aggregation." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276197.
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The self-assembly of amyloidogenic proteins to form cytotoxic species that give rise to brain deterioration underlies numerous neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Increasing evidence indicates that it is the rare, low-molecular-weight species (oligomers) rather than the more abundant high-molecular-weight fibrils of certain proteins that are the most cytotoxic in several neurodegenerative diseases. However, these species have proven difficult to study using traditional methods due to their transient nature and the heterogeneity of aggregation mixtures. In this thesis, I describe my work to develop advanced methods where I combine single-molecule and ensemble fluorescence techniques with microfluidic strategies to enable the study of protein aggregation, spanning small, transient oligomers to large, insoluble aggregates. In Chapter 1 I give an overview of the biological context and relevance of this work, including the background of neurodegenerative disease, amyloidogenic aggregation and key proteins involved. I then briefly review fluorescence microscopy techniques and the field of microfluidics. In Chapter 2 I describe how complex microfluidics can be integrated with single-molecule confocal techniques to provide a highly sensitive method to continuously probe protein aggregation in vitro. I show, for the first time, that the dilution of aggregating mixtures may be automated, by up to five orders of magnitude, down to the picomolar concentrations suitable for single-molecule measurements. By incorporating this microfluidic dilution device I greatly improve the temporal resolution of the technique and facilitate the observation of more transient species through the ability to rapidly dilute and take fluorescence measurements of samples. In Chapter 3 I overcome the need for in situ labels to monitor amyloidogenic aggregation using single-molecule confocal microscopy. I describe my work to adapt the single-molecule confocal technique to achieve the ultrasensitive detection of individual aggregate species under flow without covalently-attached labels. I have demonstrated the ability of this new method to monitor the aggregation of label-free amyloidogenic proteins using extrinsic labels ex-aggregation, opening the way for biological samples to be probed in a high-throughput manner. In Chapter 4 I describe my work to combine the high precision of confocal microscopy with a microfluidic device developed to directly characterise the sizes and interactions of biomolecules in the continuous phase. By monitoring the spatial and temporal mass transport on the micron scale, the diffusion coefficient, and thus hydrodynamic radius, of species may be determined. The technique delivers much greater sensitivity for size quantification, allowing scarce and other challenging samples to be characterised, and provides significant steps towards accurate sizing for single-molecule aggregation experiments under flow. In Chapter 5 I describe my work to determine the microscopic driving force for the spatial propagation of amyloid-beta. The epifluorescence instrument I built has enabled the proliferation of aggregate species to be monitored over a macro distance on a timescale of minutes. This has greatly improved the scope of the experimental data attained, which will be used in conjunction with Monte Carlo simulations to deliver a model for the propagation of amyloid-beta in vitro. Together this thesis represents my work developing the above novel fluorescence techniques to improve their temporal and size resolution, sensitivity and adaptability to study highly complex and fundamental protein aggregation linked to neurodegenerative disease.
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Robinson, Matthew D. "A novel fluorinated probe for medical imaging." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:3f9e6bbf-bbda-45c3-9ff9-826463ff011e.
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Selvan, Nithya. "Reductionist animal models to probe protein O-GlcNAcylation." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/6034adb4-fc6a-4c47-ade6-74e0b18d4297.
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Protein O-GlcNAcylation is a reversible type of glycosylation of serine and threonine residues of nucleocytoplasmic proteins occurring in all animals examined to date. Its installation on protein substrates is carried out by O-GlcNAc transferase (OGT), and its removal by O-GlcNAc hydrolase (O-GlcNAcase or OGA). A range of proteomics studies have suggested that over a thousand intracellular proteins are O-GlcNAc modified. However, little is known of the role of O-GlcNAc on these proteins. In a search for a reductionist model with a small proteome to dissect the general global mechanisms of protein O-GlcNAcylation, I discovered the presence of functional OGT, OGA and nucleocytoplasmic protein O-GlcNAcylation in the most basal extant animal, the placozoan Trichoplax adhaerens. I show via enzymatic characterization of Trichoplax OGT/OGA and genetic rescue experiments in Drosophila melanogaster that these proteins possess activities/functions similar to their bilaterian counterparts. While Trichoplax is not currently amenable to genetic manipulation, studying protein O-GlcNAcylation in this organism has revealed the presence of OGT, OGA and O-GlcNAc in non-bilaterian animals. Together with the absence of O-GlcNAcylation in lower organisms, this suggests that OGT-dependent reversible protein O-GlcNAcylation is a metazoan innovation, which may have facilitated the rapid and complex signaling mechanisms required for the evolution of multicellular organisms. Various genetic approaches in several animal models have revealed that protein O-GlcNAcylation is required for embryogenesis. Embryonic development in a genetically tractable organism is thus a model in which the mechanisms of protein O-GlcNAcylation can be investigated. Drosophila melanogaster OGT is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the modified proteins involved, and the underlying biology linking these to embryonic development are poorly understood. One of the limiting factors towards characterizing O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. Harnessing the unusual properties of a catalytically inactive bacterial O-GlcNAcase mutant that binds O-GlcNAc sites with sub-micromolar affinity, I show that protein O-GlcNAcylation is dynamic along Drosophila embryonic development. In addition to immunoprecipitation using the anti-O-GlcNAc antibody RL2, I have used the mutant OGA probe to enrich for O-GlcNAcylated proteins from samples of embryos at various developmental stages, and using mass spectrometry, identified novel conserved O-GlcNAcylated proteins. There is evidence in the literature for some of these O-GlcNAc proteins being involved in the regulation of hox gene expression, suggesting that the lack of O-GlcNAcylation of these proteins may contribute to the homeotic phenotypes observed in ogt null Drosophila mutants. This thesis therefore lays the foundation for the investigation of the mechanisms by which protein O-GlcNAcylation of specific substrates would affect a process such as embryonic development.
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Habtemariam, Mesay. "Bioinformatics Approach to Probe Protein-Protein Interactions: Understanding the Role of Interfacial Solvent in the Binding Sites of Protein-Protein Complexes;Network Based Predictions and Analysis of Human Proteins that Play Critical Roles in HIV Pathogenesis." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/2997.
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The thesis work contains two projects under the same umbrella. The first project is to provide a detailed analysis on the behavior of interfacial water molecules at protein-protein complexes, in this case focusing on homodimeric complexes, and to investigate their effect with respect to different residue types. For that reason the homodimeric data-set, which includes high-resolution (≤ 2.30 Å) X-ray crystal structures of 252 (140 Biological & 112 Non-biological) protein complexes was chosen to explore fundamental differences between interfaces that Nature has “engineered” vs. compared to interfaces found under man-made conditions. The data set was comprised of 5391 water molecules where a maximum of 4 Å from both interfacing proteins. Our analysis is applied a suite of modeling tools based on HINT, a program for hydropathic analysis developed in our laboratory. HINT is based on the experimental measurement of the hydrophobic effect. The second project is designed to explore various means of suppressing the expression of human genes that play critical role in HIV pathogenesis. To achieve this aim, a data set of Affymetrix Human HG Focus Target Array, which measures the expression levels of HIV seronegative and seropositive individuals in human PBMCs, was analyzed with Pathway Studio 9.0 software. This work gives insight into the elucidation of the important mechanisms of human proteins interactions in HIV seropositive individuals and their implications. Hence, we found the kind and types of microRNAs that are suppressing the human genes which have great role for HIV replication in a cell.
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CUNATI, DIANA. "Ruolo dei lipid raft nel metabolismo della proteina prionica." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/27001.
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The prion protein (PrP) is a GPI-anchored protein primarily concentrated in neuronal cells. Under certain conditions, the innocuous cellular form of this protein, PrPC, can convert into the lethal scrapie isoform, PrPSc, which can aggregate with other PrP molecules and exert its neurotoxic activity.
The structure of PrPC consists of two domains: an N-terminal, glycosylated, flexible disordered domain which is capable of binding copper and a C-terminal α-helical domain.
In contrast, PrPSc is enriched in β-sheet structures and characterized by its poor solubility in non-denaturing detergents, propensity for aggregation, partial resistance to proteinase K digestion.
The conversion of PrPC into PrPSc occurs in particular regions of the cell membrane, enriched with cholesterol and glycosphingolipid, called lipid rafts; these microdomains are thought to play a crucial role both in physiological functions and in the alternative folding of the prion protein.
In addition, it’s known that:
-PrPC can be cleaved at the 110/111 peptidyl bond to produce a C-terminal fragment, C1, which remains membrane bound and a N-terminal fragment, N1, released in the extracellular space. C1 fragments can’t be converted to the scrapie isoform;
- in cell cultures, ADAM10 and ADAM17 were shown to be responsible for this processing and their activation seems PKC-dependent.
The aim of our project is to establish if the alteration of cell lipid composition can modify the membrane distribution of the prion protein within rafts or non-raft regions and promote the activity of disintegrins such as ADAM10/17 upon the prion protein.
For this reason, granule cells, from the cerebella of 8-day-old rats, were incubated after 8 days in culture with GM1 or GD1a or GT1b for 4 hours at 37°C or with GM1 for 4 hours at 4°C. Detergent resistant fractions, containing lipid rafts, were isolated and proteins in all gradient fractions were separated and analyzed by EF/WB with specific antibodies.
After cell treatments with exogenous gangliosides, a good percentage of them was found in lipid rafts; immunoblotting analysis with specific antibodies showed a significant reduction in the amount of proteins, normally localized in lipid rafts, after incubation with GT1b.
The incorporation of this ganglioside, characterized by a remarkable steric encumbrance, might be responsible for lipid rafts destabilization and proteins redistribution toward non-raft regions.
Another possibility is that GT1b incorporation reduces the number of lipid rafts on the cell membrane.
Immunoblotting analysis with three different anti-PrP antibodies showed that this protein is not selectively located in lipid rafts but it is also distributed in several intracellular compartments.
Cell treatments with GM1 or GD1a at 37°C for 4 hours were not able to promote PrPC cleavage at the 110/111 peptidyl bond; cell incubation with GM1 seemed able to induce a conformational change of the prion protein toward a “simil-scrapie” isoform, partially resistant to classical denaturation protocols. Further studies are in progress to fully demonstrate that GM1-PrP interaction results in this conformational change.
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D'Angelo, Anna. "Functional characterization of the human PRUNE protein : implications in cancer." Thesis, Open University, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406475.
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Shadmehr, Mehrdad, and Mehrdad Shadmehr. "Design and Synthesis of Triazabutadiene-based Fluorogenic Probes for Tyrosine Specific Labeling of Proteins." Diss., The University of Arizona, 2018. http://hdl.handle.net/10150/626757.
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Chemical labeling is an important tool for understanding protein structure and function. Biological research often requires the use of molecular labels that are covalently attached to facilitate detection or purification of the labeled protein and its binding partners. Although the number of probes have been developed for labeling of specific residues of proteins is substantial, there is still a need for new reagents with better reactivity, and selectivity. Moreover, these chemical probes should be able to label the protein of interest under mild biologically relevant conditions.
Aryl diazonium salts have been utilized for selective modification of tyrosine residues. However, most diazonium compounds need to be generated in situ under strongly acidic conditions due to their instability1. Our group has previously shown that triazabutadienes can be used as precursors that can generate diazonium under mild acidification2 or
photo-irradiation3. Current reported systems for bioconjugation of tyrosine require an additional step for fluorescent labeling4. To address this issue and reduce background fluorescence that is associated with fluorescent labeling, coumarin triazabutadiene-based fluorogenic probes were synthesized and tested for tyrosine specific labeling of proteins under mild acidic condition or photo-irradiation.
Furthermore, a coumarin triazabutadiene-based cross-linker was synthesized with an azide functionality that can be used to attached the coumarin triazabutadiene warhead onto the surface of a protein. Upon the activation of the triazabutadiene group, by light or lowering the pH, this system can generate a coumarin diazonium salt on the surface of the protein. Such a system can find application in the study of protein-protein interactions and virus-protein interactions.
A cyclooctyne triazabutadiene was synthesized to attach a cyclooctyne group on the tyrosine residues of proteins in biologically relevant pH, and 3-azido 7-hydroxy coumarin was made as a fluorogenic partner of the cyclooctyne triazabutadiene. It was demonstrated that this system can label tyrosine residue followed by a copper-free click reaction with the azido coumarin fluorophore. This system has been tested on model proteins and can be consider as one the first fluorogenic triazabutadiene systems that can be utilized for labeling of tyrosine under mild conditions.
In conclusion, this dissertation demonstrates progress in developing fluorescent and fluorogenic triazabutadienes systems for labeling of tyrosine residues of proteins as well as fluorophore triazabutadiene cross-linker that can be used for studying protein-protein interaction, and virus-protein interactions. These systems offer a convenient tool to those wishing to study proteins, protein-protein interactions, and virus-protein interactions.
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Dehon, Patricia M. "Localization and Mutational Analysis of the Nuclear and Aggregation-Prone Ime4 Protein in Saccharomyces cerevisiae." ScholarWorks@UNO, 2012. http://scholarworks.uno.edu/td/1567.
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In Saccharomyces cerevisiae, Ime4 is a protein that is induced during meiosis and has a primary role in regulating sporulation in starving diploids. One function of Ime4 is methylation of adenosine residues within mRNA transcripts. Recent studies have shown Ime4 to be induced in haploids during the mating response, although its role in mating has not been determined. In this report, I identify the subcellular localization of Ime4 during the mating response through treatment with alpha factor. A plasmid containing IME4-GFP under the control of the medium strength promoter CYC1 was created in order to express the protein in a controlled manner. Lastly, mutational analysis was conducted to determine which regions of the protein were necessary for its nuclear localization, aggregation, and sporulation function.
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Egleton, James Edward. "Small molecule colorimetric and fluorescent probes for specific protein detection." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:0a1a1c80-8055-491a-920a-3e17f7919e93.
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This thesis describes the design, synthesis, analysis, mechanistic evaluation and optimisation of small molecule probes for the specific detection of proteins, focusing on the target protein human arylamine N-acetyltransferase type 1 (HUMAN(NAT1)) and its murine homologue, mouse arylamine N-acetyltransferase type 2 (MOUSE(NAT2)). The HUMAN(NAT1) gene is reported to be one of the most highly overexpressed genes in estrogen-receptor-positive (ER+) breast tumours, leading to its potential use as both a novel diagnostic biomarker and a novel therapeutic target for this disease. Chapter 1 reviews the literature on optical methods for the specific detection of a protein target, exploring strategies both based on biosensors and on chemical probes, before introducing the arylamine N-acetyltransferases as a family of enzymes. In Chapter 2, a family of naphthoquinone inhibitors of HUMAN(NAT1) are introduced, which undergo a colour change from red to blue upon binding specifically to the enzyme. The mechanism of this colour change, a proton transfer-mediated process, is discussed via the synthesis, pharmacological and colorimetric evaluation of close analogues of the hit compound lacking a key acidic sulfonamide-NH proton. During these studies, it was found that direct O-methylation of a sulfonamide is possible under certain conditions; such a reaction has not previously been reported. Furthermore, upon heating in polar solvents the O-methylated sulfonamide was observed to undergo rearrangement, and the mechanism of this process is investigated via NMR and kinetic studies. In Chapter 3, the design, synthesis and evaluation of HUMAN(NAT1) inhibitors with improved pharmacological and colorimetric profiles over the initial hit are described. From this optimisation, structure-activity relationships and an in silico model of interactions between the inhibitors and enzyme are evaluated. Testing of these compounds in cellular environments, however, exposes some limitations of this approach, notably the lack of sensitivity of the probes when dosed at low concentrations in cellular samples. In order to overcome this limitation, in Chapter 4 fluorescent analogues of the hit compound are designed and synthesised. Initial compounds developed in this series possess promising properties, but each compound generated suffers from either a low fluorescent intensity, lack of a pH-dependent switch in fluorescence or a low fluorescence excitation wavelength, which overlaps with those of tryptophan or tyrosine residues in proteins. Insights into the mechanism of molecular fluorescence and application of some simple quantum mechanical principles, however, lead to the design of a species which possesses all the required properties. The fluorescent emission intensity of this probe correlates linearly with [MOUSE(NAT2)] in E. coli cell extracts, and can quantify as little as 0.64% MOUSE(NAT2) in the samples; furthermore, the probe is capable of unambiguously detecting HUMAN(NAT1) within a cell extract from the ER+ breast cancer cell line ZR-75-1; future work on this probe may therefore enable its clinical use in improved early diagnosis of breast tumours. This study also represents, to the best of our knowledge, the first ever example of a small molecule, non-covalent probe capable of quantifying the concentration of a target protein in cellular extracts. In Chapter 5, the series of naphthoquinone probes is further optimised in order to study the roles of HUMAN(NAT1) in a cellular environment. Firstly, structure-activity relationships are utilised to design inhibitors with improved physical properties such as aqueous solubility and cell membrane permeability, in order to test the effect of HUMAN(NAT1) inhibitors in tumour cell models, which could have implications for the future use of a HUMAN(NAT1) inhibitor as a therapeutic agent in oncology. Secondly, the effect of the cofactor folic acid on the function and activity of HUMAN(NAT1) is explored. Finally, in Chapter 6, the conclusions of this study are outlined and a hypothesis as to how the concepts developed in this thesis might be applied to alternative, more ubiquitous biological targets is discussed, paving the way for future investigations.
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Caufield, J. Harry. "Interactomics-Based Functional Analysis: Using Interaction Conservation To Probe Bacterial Protein Functions." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4580.
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The emergence of genomics as a discrete field of biology has changed humanity’s understanding of our relationship with bacteria. Sequencing the genome of each newly-discovered bacterial species can reveal novel gene sequences, though the genome may contain genes coding for hundreds or thousands of proteins of unknown function (PUFs). In some cases, these coding sequences appear to be conserved across nearly all bacteria. Exploring the functional roles of these cases ideally requires an integrative, cross-species approach involving not only gene sequences but knowledge of interactions among their products. Protein interactions, studied at genome scale, extend genomics into the field of interactomics. I have employed novel computational methods to provide context for bacterial PUFs and to leverage the rich genomic, proteomic, and interactomic data available for hundreds of bacterial species.
The methods employed in this study began with sets of protein complexes. I initially hypothesized that, if protein interactions reveal protein functions and interactions are frequently conserved through protein complexes, then conserved protein functions should be revealed through the extent of conservation of protein complexes and their components. The subsequent analyses revealed how partial protein complex conservation may, unexpectedly, be the rule rather than the exception. Next, I expanded the analysis by combining sets of thousands of experimental protein-protein interactions. Progressing beyond the scope of protein complexes into interactions across full proteomes revealed novel evolutionary consistencies across bacteria but also exposed deficiencies among interactomics-based approaches. I have concluded this study with an expansion beyond bacterial protein interactions and into those involving bacteriophage-encoded proteins.
This work concerns emergent evolutionary properties among bacterial proteins. It is primarily intended to serve as a resource for microbiologists but is relevant to any research into evolutionary biology. As microbiomes and their occupants become increasingly critical to human health, similar approaches may become increasingly necessary.
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Thompson, Cherrie Delos Santos. "Using the potent Pasteurella multocida toxin to probe G-protein signalling." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/using-the-potent-pasteurella-multocida-toxin-to-probe-gprotein-signalling(b9692597-dd37-4fbc-ba0d-00691809538e).html.
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The heterotrimeric G-proteins are critically important intracellular signalling molecules that regulate fundamental processes in cellular homeostasis. A unique bacterial toxin, Pasteurella multocida toxin (PMT) modifies 3 of the 4 families of G-proteins (Gq, Gi, and G12) by deamidation which leads to a plethora of downstream signalling. PMT induces drastic morphological changes such as loss of adherence to the matrix, foci and stress fibre formation in Swiss 3T3 and Vero cells. PMT is mitogenic for many cell types but not all, and the work reported in this thesis aims to compare two cell lines that respond differently to PMT. Swiss 3T3 and Vero cells were treated with different concentrations of PMT to determine the effects on cell proliferation, cytoskeletal reorganisation and cell death. PMT induced prominent cytoskeletal changes, mitogenic and anti-cell death responses in Swiss 3T3 cells while delayed cytoskeletal changes with no evidence of mitogenicity and cell death were observed in Vero cells. PMT modified G-proteins at different times in Swiss 3T3 and Vero cells. The PMT-induced anti-cell death response in Swiss 3T3 cells was dose-dependent while the delayed cytoskeletal response in Vero cells was linked to the late PMT-mediated G12 activation. The amino acid sequence of Gα12 differed in the two cell types – the G12 subunit in Vero cells is missing N-terminal cysteine residue, which may have contributed to the differences. Gq/11 signalling is active and sustained in Swiss 3T3 cells, but not in Vero cells. Gβγ may have inhibited adenylyl cyclase activity so it is unknown whether Gi signalling is active and sustained over the 3-day period as the forskolin-stimulated cAMP level decreased in both serum-starved untreated and PMT-treated cell lines. In summary, I have identified changes in both the primary effect of PMT on the two cell types and also on downstream signalling that is likely to reflect the differential cell response.
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Quintyn, Royston Samuel. "Applying Tandem Mass Spectrometry coupled with Ion Mobility to probe the Structure of Non-Covalent Protein Complexes and their Interactions with Ligands, Peptides and other Proteins." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437495530.
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Silva, Kássya Mycaela Paulino da. "Avaliação do Nível Basal e do Controle Barorreflexo da Atividade do Sistema Nervoso Simpático da Prole de Ratos que Sofreram Desnutrição Proteica Durante a Gestação e a Lactação." Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/16439.
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FACEPE
Estudos buscam esclarecer a relação entre desnutrição proteica em fase crítica do desenvolvimento e surgimento de hipertensão arterial na vida adulta, entretanto os mecanismos que participam deste processo ainda não são totalmente conhecidos. Neste estudo investigamos as repercussões da desnutrição proteica perinatal (gestação e lactação) sobre o nível basal e o controle barorreflexo da atividade do sistema nervoso simpático da prole. Ratas prenhes foram alimentadas com dieta hipoproteica (8% de proteína, grupo experimental) ou com dieta normoproteica (17% de proteína, grupo controle) durante o período perinatal. Após o desmame, a prole de ratos machos recebeu dieta padrão para animais de laboratório e os estudos funcionais foram realizados aos 90 dias de vida. Todos os protocolos experimentais foram aprovados pelo comitê de ética em utilização animal da UFPE (processo nº 23076.016256/2013-83). Para registro direto das variáveis hemodinâmicas e infusão das drogas, foram implantadas cânulas na artéria e veia femoral, respectivamente. Os níveis de atividade autonômica foram estimados através de métodos lineares e não lineares de análise da variabilidade da pressão sistólica e intervalo de pulso em animais conscientes e anestesiados, enquanto a eficiência do mecanismo barorreflexo em regular a pressão arterial foi avaliado através de métodos indiretos de sequência espontânea e de forma induzida mediante infusão de fenilefrina e nitroprussiato de sódio. Além disso, foi realizado implante de eletrodo bipolar na cadeia simpática em nível L3-L5 para registro direto da atividade elétrica do nervo lombar em animais anestesiados. Os resultados demonstram que ratos submetidos à desnutrição proteica perinatal apresentam desbalanço autonômico para o coração, com aumento do componente de baixa frequência no espectro da pressão sistólica e intervalo de pulso, aumento do índice simpatovagal para o coração e aumento dos padrões 0V, relacionados ao tônus simpático cardiovascular. Além disso, observamos a integridade do controle barrorreflexo da pressão arterial em todas as doses avaliadas, uma vez que os animais não anestesiados submetidos à restrição proteica apresentaram sensibilidade barorreflexa semelhante aos controles. Em condições de anestesia, o registro direto do nervo lombar em ratos desnutridos não foi diferente dos seus pares, demonstrando que o controle barorreflexo da atividade nervosa simpática lombar está preservado neste modelo experimental. Esses achados apontam que o desenvolvimento da hipertensão arterial na prole submetida à desnutrição proteica perinatal não está relacionada à disfunção do barorreflexo em animais conscientes e anestesiados. Sugerimos que outros mecanismos centrais e periféricos possam estar envolvidos no surgimento e manutenção dos níveis elevados de pressão arterial neste modelo experimental.
Studies seek to clarify the relationship between protein malnutrition in critical stage of development and emergence of hypertension in adulthood, however the mechanisms involved in this process are not yet fully known. We investigated the effects of perinatal protein malnutrition (pregnancy and lactation) on the baseline and the baroreflex control of the sympathetic nervous system activity on the offspring. Pregnant rats were fed a low protein diet (8% protein, experimental group) or normal protein diet (17% protein, control group) during the perinatal period. After weaning, the male offspring of rats received standard diet to laboratory animals and functional studies were performed at 90 days of life. All experimental protocols were approved by the Ethics Committee on Animal use of UFPE (process n° 23076.016256 / 2013-83). For the direct recording of hemodynamic variables and for the infusion of drugs, cannulas were implanted in the femoral artery and vein, respectively. The autonomic activity levels were estimated by linear and nonlinear methods of analysis of the variability of systolic and pulse interval in conscious and anesthetized animals. The efficiency of the baroreflex mechanism in regulating the blood pressure was evaluated by indirect methods of sequence and form spontaneously induced by phenylephrine and sodium nitroprusside infusion. We also performed bipolar electrode implantation in the sympathetic chain at L3-L5 level for direct recording of the electrical activity of the lumbar nerve in anesthetized animals. The results show that rats with perinatal protein malnutrition have autonomic imbalance to the heart, with increased low frequency component in the spectrum of systolic and pulse interval, increased sympathovagal index to the heart and increase the standards 0V, related to the tone cardiovascular friendly. In addition, we observe the integrity of baroreflex control of blood pressure in all evaluated doses, since unanesthetized animals submitted to protein restriction had baroreflex sensitivity similar to controls. In anesthesia conditions, the record of straight lumbar nerve in malnourished rats was not different from their peers, demonstrating that the baroreflex control of lumbar sympathetic nerve activity is preserved in this experimental model. These findings indicate that the development of hypertension in offspring submitted to perinatal protein malnutrition is not related to the baroreflex dysfunction in conscious and anesthetized animals. We suggest that other central and peripheral mechanisms may be involved in the emergence and maintenance of high levels of blood pressure in this experimental model.
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Rodrigues, Mariane Augusta Domingues. "Avaliação da atividade e resistência à clivagem proteolítica de L-asparaginases recombinantes obtidas por reação em cadeia da polimerase propensa a erro." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-24082016-163433/.
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A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas.Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.
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Laos, Roberto. "Protein directed evolution." Revista de Química, 2012. http://repositorio.pucp.edu.pe/index/handle/123456789/99875.
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Evolución dirigida de proteínas: La evolución dirigida es una técnica que nos permite explorar funciones enzimáticas que no son requeridas en el ambiente natural. Esta técnica, simula procesos genéticos naturales y de selección. Esta estrategia se utiliza cuando un diseño racional es muy complicado. Consiste en una repetición de ciclos de diversificación y selección que llevan a la acumulación de mutaciones benéficas. Aquí se presenta dos ejemplos de evolución dirigida con los cuales se ha trabajado directamente: la ADN polimerasa del organismo Thermus aquaticus usada comúnmente en PCR, y la proteína LacI que regula la expresión de genes usados para el metabolismo de lactosa en E. Coli.Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.
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Clark, Peter George Keith. "Discovery of epigenetic probes against the bromodomain family of proteins." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:a589d1f3-c8f6-4219-a10b-79099eafe1c8.
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Chemical probes are necessary for elucidating the biochemical roles of proteins. Bromodomains are protein-interaction modules found in a family of proteins implicated in the epigenetic regulation of transcription; however, the individual roles remain unknown for many bromodomain proteins, without potent and selective ligands available to assist in their study. From lead compounds, a structure-based drug discovery program was to be explored with the use of biophysical assays and appropriate chemical methods to expediate development of probes against a number of these proteins. A fragment lead against BRD4 was developed into PNZ5, a potent (KD 5 nM) BRD4 probe with a high ligand efficiency. Although enantioselective syntheses and the use of an alternative synthetic route were unsuccessful, PNZ5 showed cytotoxic activity against gastric cancer cell lines that had proved resilient to existing anticancer agents. Optimisation of a lead compound against BRD9 resulted in the development of LP99, the first reported BRD7/9 probe, that was potent (BRD9 KD 99 nM, BRD7 KD 909 nM), selective amongst bromodomain proteins and active in cells. An enantioselective synthesis was performed using chiral organocatalyts and LP99 was used to identify a previously unknown role of BRD7/9 in the regulation of inflammatory processes. Research is ongoing to assess further biochemical roles of these proteins with LP99. Arising from a more potent lead against BRD9, a series of structurally related compounds were synthesised to explore SAR around this ligand, however no improvement on the affinity of the lead was realised. Finally, based on disclosed lead structures against PCAF, a series of compounds were synthesised to replicate their activity. A number of important binding interactions were assessed and a lead structure was identified (KD 1 μM). Development is ongoing to progress this lead into the first reported PCAF probe.
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Stofella, Michele. "Hydrogen deuterium exchange: methods to probe protein dynamics at single residue resolution." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amslaurea.unibo.it/21242/.
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The purpose of this work is to provide computational methods to fingerprint protein dynamics probed by hydrogen deuterium exchange mass spectroscopy (HDX-MS). Hydrogen deuterium exchange consists in the spontaneous exchange of amide hydrogens of amino acids with deuterium contained in solution. The consequent increase in mass of the protein can be monitored by mass spectroscopy. Moreover, the exchange rate (or protection factor) provides a parameter probing protein dynamics at single residue resolution. The ExPfact algorithm is a computational method implemented to extract fine-grained information out of coarse-grained HDX-MS experimental data. The method is validated through a comparison with protection factors estimated from HDX-NMR measurements probing the mouse prion protein. Also, a second application studying glycogen phosphorylase shows how structural changes between different states of the same protein can be detected at amino acidic resolution. Furthermore, fine-grained information extracted by ExPfact is coupled with a back-exchange correction to reproduce experimental spectra, suggesting that the information encoded in the centroids of the spectra is sufficient to characterize experimental data. Last but not least, an existing structural model connecting the structure of a protein to its protection factors is discusses and improved via the introduction of a dependence on the electrostatic potential of the protein.
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Huisstede, Jurgen Hendrikus Gerhardus. "Scanning probe optical tweezers a new tool to study DNA-protein interactions /." Enschede : University of Twente [Host], 2006. http://doc.utwente.nl/55834.
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Chen, Changsheng Verfasser], and Maximilian [Akademischer Betreuer] [Ulbrich. "Fluorescently labeled DNA probe in STORM imaging and single-molecule protein labeling." Freiburg : Universität, 2019. http://d-nb.info/1187658545/34.
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Chen, Changsheng [Verfasser], and Maximilian [Akademischer Betreuer] Ulbrich. "Fluorescently labeled DNA probe in STORM imaging and single-molecule protein labeling." Freiburg : Universität, 2019. http://d-nb.info/1187658545/34.
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Khan, Md Ahad Ali. "Molecular weight information from protein-ligand complexes to probe natural product interactions." Thesis, Griffith University, 2017. http://hdl.handle.net/10072/371908.
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This project presented a strategy to investigate natural product binders of protein targets based on their molecular weights. The molecular weight of the binders was determined from non-covalent protein-natural product complexes, detected by native mass spectrometry screening of natural product libraries including extracts, fractions and pure compounds. Human (Homo sapiens) calcium binding protein S100A4 (apo and calcium bound forms), mouse (Mus musculus) T cell/transmembrane, Ig, and mucin (TIM) protein 3 and human T-cell immunoreceptor with Ig and ITIM domains precursor protein (TIGIT) were investigated in this project. For functional association analysis of the proteins, protein-protein interaction (PPI) networks were constructed. The PPI network of S100A4 demonstrated that it is an important target in different types of cancers, such as breast cancer, colorectal cancer, bladder cancer, esophageal cancer, non-small cell lung cancer, gastric cancer, medulloblastoma, pancreatic cancer, prostate cancer, thyroid cancer and colon cancer. It is also a potential target in osteoarthritis, rheumatoid arthritis, pannus formation and joint destruction. TIM3 has functional association in regulatory immune processes, such as regulation of autoimmunity and anti-tumour immunity. TIGIT is an important target in autoimmune diseases caused by viral, bacterial and protozoal infections, and macrophage-mediated inflammatory diseases. It is a good target for the development of immuno-oncology combination therapies. SiteMap program was used for structure-based identification of druggable binding sites in S100A4 (apo state), S100A4-Ca2+ (calcium bound state), TIM3 and TIGIT. SiteMap scoring function, Dscore defines ‘druggability’ as a quantitative estimation of binding sites. Based on Dscore, 6 druggable binding sites were identified in apo S100A4, 6 sites in calcium bound S100A4 (S100A4-Ca2+), 2 sites in TIM3 and 5 sites in TIGIT. The druggability of a binding site was estimated as the sum of contributions from the pocket enclosure, pocket size, and the balance between hydrophobic and hydrophilic character of the binding site. The results showed that size and enclosure of a pocket has direct and proportional correlation to druggability. However, the influence of pocket enclosure on druggability was less significant than that of pocket size. The druggability of a binding site was found highly correlated to hydrophobicity of the pockets. Electrospray ionization Fourier transform mass spectrometer (ESI-FTMS) was used for direct screening of natural products. Molecular weights of hits were determined from protein-natural product complexes. Based on molecular weight, natural product binders were classified in three chemical subspaces, such as drug-like compounds with molecular weight <500 Da (RO5), lead-like compounds with molecular weight <300 Da (RO3), and beyond the ‘rule of 5’ (bRO5) for the compounds with molecular weight > 500 Da. In this project, natural product extracts and fractions were screened against four proteins including S100A4, S100A4-Ca2+, TIM3 and TIGIT. Considering the molecular weight as identifier, natural products were categorised as unique hits (binding to one protein) and common hits (binding to more than one protein). A hit detected in multiple extracts or fractions could be the same compound or different compounds with the same mass. For classification, the hits with identical molecular weight, detected in different biota were considered as the same compound. In native MS screening of extracts, 75 unique hits were detected in 86 extracts, obtained from 42 genera. Some unique hits were detected in multiple extracts. Twelve unique hits were lead-like (MW<300 Da), 36 hits were drug-(MW<500 Da) and 41 hits were beyond the rule of five (MW>500 Da). Eight unique hits were detected binding to S100A4, 38 hits to S100A4-Ca2+, 22 hits to TIM3 to and 21 hits to TIGIT. Eighteen common hits were detected in 73 extracts, obtained from 37 genera. Among them, 1 common hit was lead-like, 6 hits were drug-like and 12 hits were beyond the rule of five. Ten common hits showed binding to S100A4, 11 hits to S100A4-Ca2+, 11 hits to TIM3 and 12 hits to TIGIT. In fraction screening, 46 hits were detected in the extracts, from 15 genera. The highest number of hits was detected in fraction 3 (16 hits) and the lowest number of hits was from fraction 5 (5 hits). Six hits were detected in fraction 1, 11 hits from fraction 2, and 9 hits were from fraction 4. Twenty-three unique hits were detected in 24 extracts, obtained from 11 genera. Among them, 1 unique hit was lead-like, 13 hits were druglike and 9 hits were beyond the rule of five. Four unique hits were detected to bind to S100A4, 4 hits to S100A4-Ca2+, 7 hits to TIM3 and 8 hits to TIGIT. Fourteen common hits were detected in 24 extracts, obtained from 11 genera. Among them, 4 common hits were drug-like and 10 hits were beyond the rule of five. The active extracts were analysed by liquid chromatography high resolution mass spectrometry. Common hits from different biota showed similar or the same retention time in a C18 column. Molecular formula analysis showed that a common hit from different biota possesses the same molecular formula. Following mass-guided isolation and NMR-guided structure elucidation, the common hits, NP_564, NP_358, NP_594, NP_376, NP_434, NP_592 and NP_610 were identified as apigenin 6-C--D-glucoside 8-C--L-arabinoside, sweroside, 4',5-dihroxy-7-methoxyflavanone-6-C-rutinoside, loganin acid, 6-C-glucosylnaringenin, biochanin A 7-O-rutinoside and quercetin 3-Orutinoside, respectively. Schrödinger extra precision docking, Glide-XP was used for molecular docking of protein-natural product complexes. Binding mode of the common hits in druggable binding sites of the proteins varied with the structural features of the compounds and binding pockets. Binding affinity of four flavonoid glycosides (structural analogues) to the proteins were estimated from the standard state free energy of complex formation. All of the compounds showed the highest affinity to TIGIT and the lowest affinity to S100A4-Ca2+. S100A4-Ca2+ and TIM3 showed similar affinity for the compounds. For rapid detection of selective natural product binders, a combined screening strategy including structure-based homology search by Spot-ligand 2 and native mass spectrometry by ESI-FTMS were used. NP_204 ([2-(5-methoxy-1H-indole-3- yl)ethyl](methyl)amine) showed selective binding to S100A4, NP_217 (4-hydroxy-1- (1H-indol-3-yl)-3-methylbutan-1-one) to S100A4-Ca2+ and NP_162 (3-(1- methylpyrrolidin-2-yl)pyridine) to TIGIT.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Mosconi, Francesco. "Fluctuations in biological molecules : tools to probe mechanical and structural properties of DNA and proteins." Paris 6, 2008. http://www.theses.fr/2008PA066344.
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La plupart des réactions chimiques dans la cellule sont catalysées par des protéines dont la configuration tridimensionnelle assure leur spécificité fonctionnelle. L'information génétique nécessaire à la cellule pour assurer son fonctionnement est contenue dans l'ADN. La relation entre les propriétés structurelles et fonctionnelles d'une protéine est encore mal connue et une connaissance détaillée des propriétés mécaniques de l'ADN est indispensable si l'on veut comprendre les interactions ADN-protéines. Nous avons développé des techniques permettant de mesurer quantitativement des grandeurs d'intérêt biologique comme le module de torsion de l'ADN. Nous avons mis au point un dispositif expérimental visant à explorer, par fluorescence, la dynamique des fluctuations d'activité d'une enzyme isolée ainsi qu’un second dispositif expérimental basé sur une technique de pinces magnétiques. Nous montrons que l'on peut appliquer à l'ADN un couple calibré en même temps qu'une force externe.
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Mosconi, Francesco. "Fluctuations in biological molecules: tools to probe mechanical and structural properties of DNA and proteins." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3426746.
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The traditional boundary between hard sciences (physics and mathematics) and soft sciences (chemistry and biology) is progressively fading away as the complexity inherent in the biological world is understood and mapped out thanks to a joint attack on two fronts. On the one side more quantitative experiments allow to investigate the details of the atomic structures of biological molecules and to measure with greater precision the laws of interaction among dierent molecules; on the other side, the massive introduction of information technology in the management and catalogation of the multitude of molecular components found inside a cell is allowing to gain deep insights in the complex dynamic equilibrium that regulates the network of interactions amog different molecules. The work described in this thesis concerns the first side of the battlefield: the development of new techniques to allow quantitative measurements of biologically relevant quantities.
The work consisted in the design, construction and validation of three different experiments dealing with proteins and DNA mechanics. Key components of the cellular microcosmos, DNA and proteins are large macromolecules that constantly interact and accomplish most of the tasks needed by the cell to survive. The first part of the thesis summarises the known properties of these molecules and introduces the motivations driving the designed experiments.
Proteins catalyse chemical reactions in the cell and their threedimensional conguration gives each of them its specic function. The connections between structural and chemical properties of a protein are a subject largely unexplored.The second part of the thesis describes an experiment based on single molecule fluorescence microscopy designed to explore the dynamics of fluctuations of catalytic activity of a single enzyme.
The experiments described in this part have not yet given the hoped results. However part of the preliminary considerations done when building these setup were used to write the article F. Mosconi et al. "Some nonlinear challenges in biology", Nonlinearity 21 (2008) T131-T147.
DNA stores the genetic information needed by the cell to accomplish its tasks, and such information must be physically stored, read, written and restored in different times during the cell cycle. The importance of a proper knowledge of its mechanical properties is fundamental if its interaction with proteins is to be understood. The third part of this thesis describes two different experiments based on the magnetic tweezers micro-manipulation technique, that attempt to measure some not yet entirely characterised mechanical properties of DNA.
The two experiments presented in this part gave interesting results. A new determination of the biologically relevant parameter C, the twist modulus of DNA was obtained developing a novel type of analysis of data collected using the standard magnetic tweezers apparatus. Also, a new type of "soft" magnetic tweezers that allows the simultaneous application of an external force and an external torque has been developed and validated to measure the torque response of a DNA molecule. The results described in this part of the thesis are summarised in two papers that are ready to be submitted.
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Canale, Vinicius 1986. "Estudo funcional e morfológico renal da prole de ratos cujas mães foram submetidas à restrição proteica gestacional : efeito do tratamento com rapamicina." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312741.
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Orientadores: José Antônio Rocha Gontijo, Flávia Fernandes MesquitaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Já está bem documentado que a dieta hipoproteica em ratos durante a gestação produz efeitos no crescimento fetal, uma persistente elevação na pressão arterial e disfunções no desenvolvimento renal da prole. Alterações na pressão arterial parecem estar relacionadas à acentuada redução no número de nefros que acaba por causar um quadro de hipertrofia e hiperfluxo nos nefros remanescentes como adaptação para equilibrar a taxa de filtração, no entanto, os mecanismos utilizados para esta adaptação culminam com o surgimento de albuminúria. Este processo acaba por causar esclerose glomerular culminando em um ciclo, comprometendo cada vez mais os nefros remanescentes. O desenvolvimento deste quadro pode levar à síndrome nefrótica e posteriormente à doença renal terminal. Acredita-se que o principal fator atuante na programação fetal neste modelo é devido à exposição exacerbada do feto aos glicocorticoides materno, o que acaba por comprometer a correto desenvolvimento e diferenciação de tecidos e órgãos, e na expressão ou atividade de uma série de receptores e enzimas. Recentemente, tem surgido a hipótese de que a atividade da mTORC poderia estar envolvida no surgimento de doenças na idade adulta neste modelo experimental. Este trabalho teve por objetivo avaliar se a inibição da mTORC através do tratamento com rapamicina em animais programados poderia ser benéfico, inibindo o surgimento de complicações relacionadas com a estrutura glomerular e como seria seu efeitos sistêmico, sobre a função renal e pressão arterial sistêmica. Ratos Wistar receberam ração com baixa proteína (6% LP) e dieta controle (17% NP) durante o período gestacional. A prole de machos foi tratada com rapamicina diluída em DMSO (5%) e administrada via intraperitoneal na dose de 1mg/kg, 3 vezes por semana, a partir da 4ª semana de vida até a 12ª semana. A aferição da pressão arterial sistólica foi realizada nas idades 8, 12 e 16. Foi observado que nos grupos que receberam rapamicina, a pressão artéria sistólica elevou-se consideravelmente em todas as idades. A avaliação da função renal foi realizada através de clearance de creatinina e lítio nas mesmas idades e observamos que durante todo o tratamento, o grupo NP que recebeu rapamicina excretou mais sódio, na porção pós-proximal do túbulo. Além disso, não houve diferença na taxa da filtração glomerular em nenhuma das idades. Quando a proteinúria foi avaliada, observamos que o grupo programado LP sem rapamicina, apresentou evolução com o passar das semanas sendo significativamente maior a partir das 12ª e 16ª semanas, no entanto, os grupos que receberam rapamicina, não apresentaram a mesma evolução, indicando preservação da estrutura glomerular. Os presentes resultados demonstram que apesar de a rapamicina ter elevado a pressão arterial em ambos os grupos, há uma indicação de que os animais programados tem um controle menos eficaz no controle da pressão arterial através da função renal. Mesmo diante da pressão elevada, a rapamicina foi capaz de inibir injúrias à barreira de filtração
Abstract: It is well established that a low protein diet in rats during pregnancy causes effects on fetal growth, a persistent elevation in blood pressure and renal dysfunction in the offspring at a later age. Alterations in blood pressure seem to be related to the marked reduction in the number of nephrons which ultimately causes an overflow and hypertrophy in the remaining nephrons in the attempt to adapt its balance on the filtration rate. However, the mechanisms used for this adaptation results in the appearance of albuminuria. This process might cause glomerular sclerosis culminating in a cycle, increasing the damage to the remaining nephrons. The development of this framework can lead to nephrotic syndrome and subsequently to ESRD. It is believed that the main factor in the fetal programming model is the fetus overexposure to maternal glucocorticoids, which ultimately compromises the proper development and differentiation of tissues and organs. Additionally, the expression and the activity of a number of receptors and enzymes is also affected. Recently, in this experimental model, there has arisen the hypothesis that the activity of mTORC could be involved in the onset of disease in adulthood. This study assess whether the inhibition of mTORC, through the treatment with rapamycin in programmed animals, could be beneficial by inhibiting the onset complications related to glomerular structure and its affect on renal function and blood pressure. Wistar female rats were fed with low protein (6 % LP) or control diet (17 % NP) during pregnancy. The male offspring were treated with rapamycin diluted in DMSO (5 %) and administered intraperitoneally at a dose of 1mg/kg, 3 times per week, from the 4th week of life until the 12th week. The measurement of systolic blood pressure was measured at 8, 12, and 16 weeks old. We noticed that in the groups treated with rapamycin the arterial systolic pressure rose considerably in all ages. The assessment of renal function was performed by creatinine clearance and lithium at the same ages as the blood pressures assessment. The NPR group that received rapamycin had an increase in sodium excretion at the post- proximal tubule during the whole treatment. Additionally, there was no difference in the rate of glomerular filtration rate at any age among the groups. When proteinuria was assessed, we found that the programmed Group LP without rapamycin, showed an increase along the weeks and as expected, the groups that have received rapamycin did not show the same trend, indicating preservation in the glomerular structure. The present results demonstrate that rapamycin caused increase in the blood pressure in both groups however, it was able still to inhibit the injury into the filtration barrier. Additionally, there is an indication that the programmed animals, has a less effective control in the blood pressure and the excretion of sodium, even when the treatment is interrupted
Mestrado
Fisiopatologia Médica
Mestre em Ciências
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McDonagh, Paul David. "Protein engineering to probe the catalytic mechanism of Alpha-class glutathione S-transferases." Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/29679.
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Glutathione S-Transferases (GSTs) are a large family of isoenzymes that catalyse the addition of glutathione to hydrophobic electrophiles. Over-expression of GSTs in tumours leads to resistance to chemotherapy drugs, thus understanding GST biochemistry is clinically important. However, the catalytic and substrate recognition mechanisms remain poorly understood.;Two rat, alpha-class GSTs, Yc1 and Yc2 have 91% homology but have specific activities of 0.01 and 13.2 nmoles/min/mg respectively for the carcinogen aflatoxin-exo-8,9-epoxide. The protein structures for each were homology modelled on the co-ordinates for alpha-class human GSTA1-1. S-aflatoxinyl glutathione modelled into the active sites identifying positions 108 and 208 as important residues. A 'knock-out' double mutant D208MY108L was made in rGST Yc2 and an 'engineered' E208DH108Y mutant was made for Yc1.;The mutations reduced rGST Yc2 activity to <0.01 nmoles/min/mg and increased rGST Yc1 activity to 0.32 nmoles/min/mg which was further used to protect a human bronchial cell line against aflatoxin B1. Modelling of S-aflatoxinyl glutathione into huGSTA1-1 suggested the same positions were important in determining its low activity for aflatoxin-exo-8,9-epoxide. The double mutant L108YM208D failed to engineer any significant activity for aflatoxin-exo-8,9-epoxide into the enzyme.;The C-terminus of huGSTA1-1 was deleted and the kinetics of the truncated enzyme determined in the presence and absence of a synthetic peptide designed to replace the helix sequence. Kcat and Km were modified for the deleted enzyme in the presence of the peptide but Kcat/Km was not, suggesting the helix plays a part in promoting productive substrate binding.;The catalytically important Tyr9 was investigated by NMR. Tyr9 is thought be responsible, in part, for the deprotonation of glutathione during catalysis and as such must have a lower pKa than tyrosine in solution. Assignment of Tyr9 in the NMR spectrum allowed its titration, confirming that the pKa of Tyr9 is shifted from 10.0 to 7.720.21.
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Sun, Li. "Advances in the use of phosphorescence spectroscopy as a probe of protein flexibility." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq30395.pdf.
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Li, Xin. "Development and Application of Chemical and Structural Biology Approaches to Probe Protein Function." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306439016.
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Worthman, Lynn-Ann D. "Surfactant protein A (SP-A) affects pulmonary surfactant morphology and biophysical properties." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0014/MQ34241.pdf.
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Sagle, Laura B. "Carbon-deuterium bonds as an infrared probe of protein dynamics, local electrostatics and folding." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3195630.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed February 28, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Shearer, Lee. "Design and synthesis of ligands to probe the interactions of retinol binding protein (RBP)." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713694.
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Retinol binding protein (RBP) is a member of the lipocalin family of proteins that is responsible for transporting vitamin A (retinol) in the blood to the target cells of the body. RBP is released from the liver as a complex with transthyretin (TTR) and the role of the complex is to prevent RBP being excreted by the kidney. RBP binds to a specific cell receptor (STRA6) which facilitates the uptake of retinol into the cell. Studies have suggested that elevated levels of serum RBP may be involved in preventing cellular responses to insulin which in turn leads to insulin resistance and eventually type 2 diabetes. This suggests that RBP could be a viable therapeutic target for treating type 2 diabetes. In this study a variety of structure-based ligand design (SBLD) methods, including virtual high-throughput screening (VFITS, using the docking tool eHiTS), de novo ligand design (using SPROUT) and shape comparison software (ROCS) were used along with X-ray crystallographic data, to design a variety of non-retinoid ligands targeted at RBP. Several small libraries of ligands were synthesised and the ligands were examined for their ability to bind to RBP along with their ability to disrupt the interactions of RBP with both TTR and STRA6. A number of ligands displayed a high binding affinity for RBP with ligands 13 and 19 being two of the most potent (KD = 200 and 343 nM respectively). Several ligands were found to disrupt the interaction between RBP and TTR with ligand 26 (EC50 = 891 nM) having the greatest effect. The study identified several ligands that disrupted the interaction between RBP and STRA6 with ligands 23 and 31 (97% and 100% respectively) having the greatest effect. Rationalisation of the results using the predicted binding poses of each ligand identified Tyr90 as being a key residue involved in making a hydrogen bond interaction with the ligand which resulted in the disruption of the RBP—STRA6 interaction. This study underlines the usefulness of SBLD for designing novel ligands that displayed a high affinity for RBP and as a consequence the ligands prevented RBP from binding to TTR and STRA6.
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Sowley, Hugh Richard. "Electron-vibration-vibration two-dimensional infrared spectroscopy as a structural probe of interactions in proteins and DNA." Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/57959.
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Modern structural biology has a number of powerful tools but despite this there are a number of problems in structural biology that these methods are unable to address. Some of these pertain to the need for large number for precise comparative structures for the drug discovery process. EVV 2DIR has the potential to fill some of these gaps, having the potential to determine molecular binding geometry. This thesis presents the first steps in exploring the potential of EVV 2DIR to be applied to the analysis of the structure of inhibitor- protein binding and presents the first EVV spectra of an inhibitor-protein complex. For the inhibitor-protein complex studied, six vibrational couplings between seven vibrational modes were identified exclusively upon complex formation due to interactions between the two molecules. Experimental spectra were compared with ab initio calculations to assign these vibrations to specific motions on both the inhibitor and protein molecules. EVV 2DIR cross peaks can be sensitive to the geometry of the interacting groups which produce them. By measuring the spectra of the inhibitor-protein complex using two different polarisation schemes, quantitative comparison between calculated and experimental spectra was made possible. This allowed for the prospect of using calculation aided EVV 2DIR to determine the structure of protein-ligand complexes to be explored. This thesis also presents the first EVV spectra of DNA. EVV 2DIR spectra were measured of duplex and G-quadruplex structures and compared with those of unstructured controls. In the absence of calculated spectra, assignments were made to some of the spectral features observed. EVV 2DIR was shown to be sensitive to the structural form of the DNA samples, containing cross peaks indicative of WatsonCrick base pairing, G-quadruplex formation and glycosidic bond conformation. The DNA spectra contained many unassigned peaks leaving open the possibility to assign many more structural indicators.
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Gantner, Matthias. "Exploring solid supported membrane based electrophysiology as an alternative platform to probe activity of membrane transport proteins." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/19682/.
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Membrane transport proteins take a pivotal role in all forms of life as they are responsible for organising traffic of ions and small molecules across the hydrophobic barrier of biological membranes. Mutations in membrane transporters can often lead to severe diseases and they often consitute drug targets. Hence, assaying function of membrane transporters is of great importance. In this project the method used for this task was mainly a relatively uncommon technique called solid-supported membrane based electrophysiology. The goal was to test this technique on targets that are challenging to investigate by more conventional methods. A first target was the TRPM2 ion channel. TRP channels are difficult to investigate because they often show a very complex activation pattern. A second target was the bacterial transition metal transporter MntH2 from Enterococcus faecalis, belonging to the SLC11 family. Transition metal transporters are generally difficult to investigate, because of the nature of their substrates. Some transition metals are redox-active and in solution they act as complexing agents. Application of solid supported membrane based electrophysiology was not successful for TRPM2, but the method was used to perform basic biophysi- II cal characterisation of MntH2. It was found that MntH2 transports a range of substrates including Mn2+, Cd2+,Co2+ and Zn2+. Ni2+ and Cu2+ were not transported and in fact inhibited manganese uptake. Interestingly, in the presence of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) electrophysiological currents were not affected. This, together with the observation from a complementary assay, that reconstituted MntH2 did not acidify the interior of vesicles loaded with pH-sensitive uorescence probes, led to the hypothesis that MntH2, contrary to common belief, is not a H+ symporter. MntH2 was attempted to crystallise and in initial screens some conditions were identified which could be a basis for optimisation in future trials.
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Nacheva, Katya Pavlova. "Development of a Bio-Molecular Fluorescent Probe Used in Kinetic Target-Guided Synthesis for the Identification of Inhibitors of Enzymatic and Protein-Protein Interaction Targets." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4376.
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Abstract
Fluorescent molecules used as detection probes and sensors provide vital information about the chemical events in living cells. Despite the large variety of available fluorescent dyes, new improved fluorogenic systems are of continued interest. The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but have remained unexplored in bioscience applications. Herein we present the identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first reported use as a donor component in Forster resonance energy transfer (FRET) systems. The FRET technique is often used to visualize proteins and to investigate protein-protein interactions in vitro as well as in vivo. The analysis of the photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large Stokes shift of 140 nm in MeOH, a very good fluorescence quantum yield in DCM (Ffl 0.61), and a high extinction coefficient ε(340) 48,400 M-1cm-1, thus ranking this molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4-difluorophenyl)-maleimide was utilized as a donor component in two FRET systems wherein different molecules were chosen as suitable acceptor components - a fluorescent quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It has been demonstrated that by designing a FRET peptide which contains the DM donor moiety and the acceptor (quencher) motif, a depopulation of the donor excited state occurred via intermolecular FRET mechanism, provided that the pairs were in close proximity. The Forster-Radius (R0) calculated for this FRET system was 36 % and a Forster-Radius (R0) of 26 % was determined for the second FRET system which contained TPP as an acceptor. The excellent photophysical properties of this fluorophore reveal a great potential for further bioscience applications. The 3,4-bis(2,4-difluorophenyl)-maleimide fluorescent moiety was also implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs) are metalloenzymes that regulate essential physiologic and physio-pathological processes in different tissues and cells, and modulation of their activities is an efficient path to treating a wide range of human diseases. Developing more selective CA fluorescent probes as imaging tools is of significant importance for the diagnosis and treatment of cancer related disorders. The kinetic TGS approach is an efficient and reliable lead discovery strategy in which the biological target of interest is directly involved in the selection and assembly of the fragments together to generate its own inhibitors. Herein, we investigated whether the in situ click chemistry approach can be implemented in the design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds, which has not been reported in the literature. In addition, we exploit the incorporation of the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide) as a potential biomarker with affinity to CA, as well as two coumaine derivatives representing a newly discovered class of inhibitors. The screening of a set of library with eight structurally diverse azides AZ1-AZ8 and fifteen functionalized alkynes AK1-AK12 led to the identification of 8 hit combinations among which the most prominent ones were those containing the coumarine and fluorescent maleimide scaffolds. The syn- and anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized, and in a regioisomer-assignment co-injection test it was determined that the enzyme favored the formation of the anti-triazoles for all identified combinations. The mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time dependent nature of the inhibition activity typical for all coumarine-based inhibitors was also observed for the triazole compounds whose inhibition constants (Ki) were determined in two independent experiments with pre-incubation times of 3 and 25 minutes, respectively. It was observed that the lower Ki values were determined, the longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles were presented as in situ generated hits which have the potential to be used as fluorescent bio-markers or other drug discovery applications.
The proteins from the Bcl-2 family proteins play a central role in the regualtion of normal cellular homeostasis and have been validated as a target for the development of anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS technology was successfully implemented in the identification of known protein-protein modulators. Optimized screening conditions utilizing a triple quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was demonstrated to be very efficient in kinetic TGS hit identification increasing both the throughput and sensitivity of this approach. The multi-fragment incubation approach was studied in detail and it was concluded that 200 fragment combinations in one well is an optimal and practical number permitting good acylsulfonamide detectability. Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and several potential hit combinations were identified. A control testing was carried out by substituting Bcl-XL with a mutant R139ABcl-XL, used to confirm that the potential kinetic TGS hit combinations were actually forming at the protein's hot spot and not elsewhere on the protein surface. Although, the synthesis of all these kinetic TGS hit compounds is currently ongoing, preliminary testing of several acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.
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Bychkova, A. V., O. N. Sorokina, P. G. Pronkin, A. S. Tatikolov, A. L. Kovarski, and M. A. Rosenfeld. "Protein-Coated Magnetic Nanoparticles: Creation and Investigation." Thesis, Sumy State University, 2013. http://essuir.sumdu.edu.ua/handle/123456789/35378.
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A novel universal approach to cross-linking of protein macromolecules on the surface of magnetite na-noparticles has been developed. The approach is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. Free radicals are locally generated on the surface of nanoparticles. Using a set of physicochemical methods, it has been proven that stable coatings composed of protein macromolecules are formed around individual nanoparticles. The proteins fixed on nanoparticles do not lose their activity as a result of adsorption and free radical modification. Fluorescent probe approach for evaluation of the native functional properties of serum albumin as a part of coating is suggested.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/35378
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Sorin, Eric J. "Biomolecular assembly in silico using massively parallel simulations to probe protein and RNA folding dynamics /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Custódio, Augusto Henrique 1983. "Estudo da denervação renal bilateral e da imunorreatividade para substância P (SP), CGRP e receptor 1 para neurocinina (NK1R) no gânglio da raiz dorsal e parede pélvica renal na prole de ratas submetidas a restrição proteica gestacional." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312740.
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Orientador: Jose Antonio Rocha GontijoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A programação fetal é um processo fisiológico que assegura, durante o desenvolvimento intrauterino, a adaptação para o mundo exterior. Ou seja, o organismo é "moldável" por estímulos durante sua formação e dependendo do insulto experimentado, existe a possibilidade de mudanças estruturais e funcionais que podem predispor o indivíduo a doenças na vida adulta. O modelo de restrição proteica, assim como outros modelos, leva a um "stress" gestacional e, segundo a hipótese de Barker, programa a prole ao desenvolvimento de doenças na vida adulta, dentre elas a hipertensão arterial. Os rins são órgãos fundamentais na manutenção do equilíbrio hemodinâmico. Mudanças morfológicas e neuroendócrinas nos rins levam a alterações hidroeletrolíticas frequentemente associadas à patogênese da hipertensão arterial. A gênese desta doença ainda não está bem descrita, por envolver alterações multifatoriais, dentre elas, modificações da atividade neural tanto central quanto periférica, que podem ser um indicativo da elevação pressórica em nosso modelo. A atividade simpática renal é um importante modulador da excreção dos eletrólitos e, quando alterada, promove maior ou menor retenção de sais, principalmente o sódio, podendo contribuir para a elevação da volemia e consequentemente da hipertensão arterial. Diversos neuropeptídeos estão envolvidos na atividade simpática renal e os níveis destes são um importante marcador na gênese da hipertensão. Dentre esses peptídeos estão a Substância P (SP), seu receptor NK1R e o Peptídeo Relacionado ao Gene da Calcitonina (CGRP). Nossos resultados mostraram redução na imunorreatividade de SP, CGRP e aumento do receptor 1 para neurocinina nos gânglios da raiz dorsal da prole de ratas submetidas à restrição proteica gestacional. Identificamos também a elevação dos níveis de CGRP na parede pélvica renal. Assim, acreditamos que haja alterações na neuromodulação da atividade aferente renal, o que pode ser um fator contribuinte para a manutenção do estado hipertensivo neste modelo experimental
Abstract: A fetal programming is a physiologic process that ensures an adaptation for external world during the intra uterine development. In this period, the organism is "moldable" by stimulus that happens during its formation, which ensures adequate phenotypes formation for different environments. Kidneys are the most important organs when it has to do with maintaining the organism hemodynamic balance and also morphological and neuroendocrine alterations, which leads to fluid and eletrolytes changes, frequently associated to arterial hypertension pathogenesis. The genesis of this disease is not well described yet. It involves multifactorial changes like the neural activity in both central as peripheral, which, in our model, may be an indicative of increased pressure. The renal sympathetic activity is an important excretion modulator of electrolytes and when amended, promotes greater or lesser retention of salts, mainly sodium, contributing to the increase in blood volume and consequently hypertension. Several neuropeptides are involved in renal sympathetic activity, and these levels are an important marker in the genesis of hypertension. Among these peptides we find substance P (SP) and its receptor NK1R, and Related Peptide Calcitonin Gene (CGRP). Our research showed reduced immunoreactivity of SP, CGRP and increased neurokinin 1 receptor in dorsal root ganglia among the offspring of rats subjected to gestational protein restriction. According to this result, we believe that there are changes in afferent renal activity neuromodulation which may be a contributing factor for maintenance of hypertension in this experimental model
Mestrado
Medicina Experimental
Mestre em Ciências
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Hanyu, Yuki. "Chemical scanning probe lithography and molecular construction." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:409308ed-4806-44fc-87c3-5c1fe8971f79.
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The initiation and high resolution control of surface confined chemical reactions would be both beneficial for nanofabrication and fundamentally interesting. In this work, spatially controlled scanning probe directed organometallic coupling, patterned functional protein immobilisation and highly localised reversible redox reactions on SAMs were investigated. Catalytically active palladium nanoparticles were mounted on a scanning probe and an appropriate reagent SAM was scanned in a reagent solution. This instigated a spatially resolved organometallic coupling reaction between the solution and SAM-phase reagents. Within this catalytic nanolithography a spatial resolution of ~10nm is possible, equating to zeptomole-scale reaction. The methodology was applied to reactions such as Sonogashira coupling and local oligo(phenylene vinylene) synthesis. By altering the experimental protocols, relating probe scan velocity to reaction yield and characterising the nanopattern, a PVP matrix model describing a proposed mechanism of catalytic nanolithography, was presented. Though ultimately limited by probe deactivation, calculations indicated that activity per immobilised nanoparticle is very high in this configuration. For biopatterning, surface nanopatterns defined by carboxylic functionality were generated from methyl-terminated SAMs by local anodic oxidation (LAO) initiated by a conductive AFM probe. By employing suitable linker compounds, avidin and Stefin-A quadruple Mutant (SQM) receptive peptide aptamers were patterned at sub-100nm resolution. The multiplexed sensing capability of an SQM array was demonstrated by reacting generated patterns with single or a mixture of multiple antibodies. The reversible redox conversion and switching of reactivity of hydroquinone-terminated SAMs was electrochemically demonstrated prior to an application in redox nanolithography. In this methodology, spatially controlled probe-induced in situ "writing" and "erasing" based on reversible redox conversion were conducted on hydroquinone terminated SAM. In combination with dip-pen nanolithography, a novel method of redox electro-pen nanolithography was designed and the method’s application for lithography was examined.
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Lee, Peter S. M. Massachusetts Institute of Technology. "Using optical tweezers, single molecule fluorescence and the ZIF268 protein-DNA system to probe mechanotransduction mechanisms." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34490.
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Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (p. 42-43).
Optical tweezers instruments use laser radiation pressure to trap microscopic dielectric beads. With the appropriate chemistry, such a bead can be attached to a single molecule as a handle, permitting the application of force on the single molecule. Measuring the force applied in real-time is dependent on detecting the bead's displacement from the trapping laser beam axis. Back-focal-plane detection provides a way of measuring the displacement, in two-dimensions, at nanometer or better resolution. The first part of this work will describe the design of a simple and inexpensive position sensing module customized for optical tweezers applications. Single molecule fluorescence is another powerful technique used to obtain microscopic details in biological systems. This technique can detect the arrival of a single molecule into a small volume of space or detect the conformational changes of a single molecule. Combining optical tweezers with single-molecule fluorescence so that one can apply forces on a single molecule while monitoring its effects via single molecule fluorescence provides an even more powerful experimental platform to perform such microscopic studies. Due to the enhanced photobleaching of fluorophores caused by the trapping laser, this combined technology has only been demonstrated under optimized conditions.
(cont.) The second part of this work will describe a straightforward and noninvasive method of eliminating this problem. The study of mechanotransduction in biological systems is critical to understanding the coupling between mechanical forces and biochemical reactions. Due to the recent advances in single molecule technology, it is now possible to probe such mechanisms at the single molecule level. The third and final part of this work will describe a basic mechanotransduction experiment using the well-studied ZIF268 protein-DNA system. An experimental assay and method of analysis will be outlined.
by Peter Lee.
S.M.
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Neely, Robert K. "The photophysical properties of 2-aminopurine and its application as a probe of DNA-protein interactions." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/12712.
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Steady-state and time-resolved fluorescence spectroscopies have been used to investigate the photophysical properties of the fluorescent DNA base analogue, 2-aminopurine (2AP). For the first time, the fluorescence decay of 2AP is shown to be biexponential. In aqueous solution, 2AP has lifetimes of 11.0ns and 13.5ns, which are shown to decrease in less polar solvents. These two lifetimes are attributed to the N7-H and N9-H tautomers of 2AP. The fluorescence responses of ten 2AP labelled DNA duplexes have been recorded. The decays are fitted by four discrete components and all are dominated by a ~100ps component, which is consistent with efficient quenching of the 2AP by electron transfer from guanine. The fluorescence response of 2AP within the duplexes is sensitive to both the nature of the bases in its immediate vicinity and the extended nucleotide sequence. 2AP labelled DNA has been used as a probe of base flipping by the M.HhaI methyltransferase enzyme. The DNA was labelled such that duplexes with the 2AP adjacent to, opposite and at the target site for base flipping have been studied. Duplexes with the 2AP outside of the M.Hhal recognition sequence have also been investigated. When 2AP is the target base for flipping its fluorescence decay shows a significant response to enzyme binding. It is shown that this change in photophysical behaviour can be used as a definitive indicator of the base flipping mechanism. A similar response is also shown for the M.TaqI base flipping enzyme. Placing the 2AP at positions adjacent to and opposite the target base for flipping demonstrate the amazing selectivity, of HhaI for its target base and demonstrate the effectiveness of the enzyme’s stabilisation of the DNA duplex during base flipping.