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1
Rubin, Jonathan. „Ion-specific and water-mediated effects on protein physical stability“. Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/47587.
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Protein aggregation and physical stability are perpetual concerns in medicine and industry. Misfolded protein can form ordered protein aggregates, amyloids, which are associated with a host of neurodegenerative diseases in mammals and control heritable traits in fungi and yeast. Industrially, amorphous aggregates reduce the efficacy of protein-based therapeutics and activity of enzymes during production and storage. This work studies ion-specific and solvent-based effects on protein physical stability. We show that ion-specificity significantly affects amyloid formation kinetics, aggregate morphology, thermostability, frangibility, and, most intriguingly, prion infectivity in vivo. Forming amyloid in chaotropic or kosmotropic solutions generates predominately weak or strong prion variants, respectively. Ion-specific effects also influenced amorphous aggregation of model proteins and antibodies. To quantify protein - protein stability/affinity, we developed a rapid and reliable diffusion-based technique. Our technique was able to resolve relative differences in colloidal stability between various saline and saccharide solutions. In all, this dissertation expands our understanding of ion-specific and water-mediated interactions with prion proteins and protein dispersions.
2
Guelev, Vladimir Metodiev. „Peptide-based polyintercalators as sequence-specific DNA binding agents /“. Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3008346.
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3
Davies, Holly Gibs. „MSY4, a sequence-specific RNA binding protein expressed during mouse spermatogenesis /“. Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/10307.
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4
Lucas, Olivier. „Molecular and systemic functions of the vertebrate-specific TATA-binding protein N terminus“. Diss., Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/lucas/LucasO0509.pdf.
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5
Rossi, Merja. „Investigating cell type specific metabolism using GFP as a reporter protein“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:0c418362-63e7-496d-9ff6-584a0c54c127.
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Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.
6
Berkes, Charlotte Amelia. „Elucidating the mechanisms by which MyoD establishes muscle-specific gene expression /“. Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5071.
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7
Xiong, Xiaoquan. „Pancreatic islet-specific overexpression of reg3β protein“. Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107823.
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Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. Reg3β (pancreatitis-associated protein, PAP; or gene expressed in hepatocellular carcinoma-intestine-pancreas, HIP) was first reported as a pancreatic secretory protein expressed in acinar cells during the acute phase of pancreatitis. Previous studies in Dr. Liu's and other research groups have demonstrated that Reg3β was specifically induced either during islet hyperplasia in the IGF-I-deficient pancreas or as a result of GLP-1-induced islet cell growth. Reg3β has been shown to play anti-apoptotic and anti-inflammatory roles during acute pancreatitis. In the present study, we generated transgenic mice with pancreatic β-cell-specific over-expression of Reg3β and investigated the effect of Reg3β in the regulation of β-cell function during streptozotocin (STZ)-induced type 1 diabetes (T1D) and diet-induced type 2 diabetes (T2D). The data presented in Chapter II demonstrated that pancreatic islet-specific overexpression of Reg3β protein protected mice from STZ-induced T1D. The RIP-I/Reg3β mice were indistinguishable from wild-type littermates in fertility, islet morphology, β-cell mass, and insulin secretion response, yet were slightly high in glucose and low in the expression of Glut2 and insulin. These transgenic mice were significantly protected from STZ-induced hyperglycemia and weight loss that were observed in the wild-type controls. To identify the molecular networks that Reg3β is involved in, a whole genome DNA microarray analysis of isolated islet RNA samples revealed more than 45 genes whose expression were markedly up- or down-regulated as a consequence of Reg3β overexpression. We further confirmed the change in several genes, including the upregulation of islet-protective osteopontin/Spp1 and acute responsive nuclear protein Nupr1/p8 by real-time PCR, Western blots and histology. These results support the potential of Reg3β in preventing STZ-induced damage by regulating expression of specific genes.In Chapter III, mice carrying Reg3β over-expression displayed worsened T2D induced by high-fat diet (HFD), as characterized by faster and more severe development of hyperglycemia, glucose intolerance and insulin resistance. Reg3β seems to exert two opposite actions in respone to HFD: further diminishing the expression of insulin and Glut2 induced by HFD, while suppressing AMPK activity and increasing p8 expression to compensate for the loss of β-cell function. Taken together, Reg3β is a putative protector that prevents STZ-induced acute damage, but unlikely an islet growth factor and unable to protect β-cells against HFD-induced T2D. The protective effect of Reg3β is likely triggered by acute stress but ineffective against chronic stress induced by HFD.Les protéines de la famille Reg sont impliquées dans la prolifération, la survie et la régénération des cellules pancréatiques β. Reg3β [aussi connue sous le nom de PAP (protéine associée à la pancréatite) ou encore HIP (gène exprimé dans le carcinome hépatocellulaire de l'intestin et du pancréas)] a été identifiée comme étant une protéine sécrétrice du pancréas et elle est exprimée dans les cellules acineuses pendant la phase aiguë de la pancréatite. Des études antérieures dans le laboratoire du Dr Liu ont démontré que la protéine Reg3β est spécifiquement induite durant l'hyperplasie des îlots pancréatiques à la suite d'un déficit de l'IGF-I ou encore à la suite d'une croissance de cellules d'îlots induite par GLP-I. Reg3β joue un rôle anti-apoptotique et anti-inflammatoire pendant la pancréatite aiguë. Dans cette étude, nous avons généré des souris transgéniques qui surexpriment Reg3β spécifiquement dans les cellules pancréatiques β afin d'étudier l'effet de Reg3β dans la régulation de la fonction des cellules β lors du diabète de type 1 (DT1) induit par la streptozotocine (STZ) ainsi que dans le cas du diabète de type 2 (DT2) induit par un régime alimentaire riche en gras. Les données présentées dans le chapitre II ont démontré que les îlots pancréatiques qui surexpriment la protéine murine Reg3β sont protégés du DT1 induit par la STZ. Les souris RIP-I/Reg3β transgéniques sont indiscernables des souris de contrôle de type sauvage en ce qui a trait à la fécondité, la morphologie des îlots, la masse des cellules β et la sécrétion de l'insuline. Cependant, une légère hyperglycémie et une faible expression de GLUT2 et de l'insuline ont été observées. Ces souris transgéniques sont considérablement protégées contre l'hyperglycémie induite par STZ et contre la perte de poids. A l'aide de puces d'ADN, une analyse d'échantillons d'ARN purifiés à partir d'îlots isolés a révélé l'existence de plus de 45 gènes dont l'expression est affectée par la surexpression de Reg3β. Nous avons également confirmé le changement d'expression de plusieurs gènes, y compris la régulation positive de l'osteopontin/SPP1 (qui protège les îlots et de la protéine nucléaire réactive aiguë p8/NUPR1) en utilisant le PCR en temps réel, le Western blot et l'histologie. Ces résultats confirment le potentiel de Reg3β dans la prévention des dommages induits par STZ en régulant l'expression de gènes spécifiques. Au chapitre III, nous avons démontré que la surexpression de Reg3β aggrave le DT2 induit par une alimentation riche en matières grasses. Ceci est caractérisé par le développement plus rapide et plus sévère de l'hyperglycémie, l'intolérance au glucose et la résistance à l'insuline. Reg3β semble exercer deux actions opposées en réponse à une diète riche en gras: 1) diminution accrue de l'expression basale de l'insuline et Glut2 et 2) suppression de l'activité de l'AMPK et augmentation de l'expression de la protéine p8 afin de compenser pour la perte de la fonction des cellules β. En résumé, Reg3β est un protecteur potentiel qui empêche les dommages induits par STZ aiguë, mais il est peu probable que ce soit un facteur de croissance des îlots pancréatiques. De plus, Reg3β est incapable de protéger les cellules β contre le DT2 induit par un régime alimentaire riche en gras. L'effet protecteur de Reg3β survient probablement en réponse au stress aigu mais il est inefficace contre le stress chronique induit par un régime alimentaire riche en gras.
8
Giorgini, Flaviano. „Functional analysis of the murine sequence-specific RNA binding protein MSY4 /“. Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10293.
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9
Hussain, Maruf Ali. „Non-specific protein interactions at model chromatographic surfaces“. Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243403.
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10
Beiersdorfer, Alex. „Site-specific Regulation of Myosin Binding Protein-C“. University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511856330493573.
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11
Georgiou, Charis. „Rational design of isoform specific ligands“. Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28713.
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Cyclophilins (Cyp) are proteins that catalyze the interconversion of trans/cis isomers of proline belonging to the peptidyl-prolyl isomerases family (PPIase). In addition to their PPIase activity, Cyps have diverse biological roles and have been implicated in a number of different diseases such as HIV-1 and HCV. Although several Cyp inhibitors have been reported in the literature, none are able to inhibit with high specificity various Cyp isoforms. To facilitate the development of isoform-specific Cyp ligands, we have pursued detailed studies of Cyp dynamics and ligand binding thermodynamics using molecular simulations, biophysical assays and protein X-ray crystallography. Research efforts were focussed on the identification of novel Cyp inhibitors using X-ray crystallographic studies and Surface Plasmon Resonance (SPR) experiments on fragments from an in-house bespoke library of small compounds. These biophysical studies revealed a number of fragments that are able to bind to diverse Cyp isoforms with high micromolar – low millimolar activity. To further examine the binding of these fragments to cyclophilins, identify interactions with the proteins and explain specificity trends from SPR and X-ray results, molecular dynamics (MD) simulations and free energy calculations were pursued. Models of apo and holo Cyps in complex with fragments that we had experimentally tested were set up using the Amber, AmberTools and FESetup software. Free energy calculations were performed using the thermodynamic integration (TI) technique with the Sire/OpenMM software. The results were analysed with custom scripts. Correlations between computed and measured binding energies, and calculated and observed binding modes were analysed to help develop guidelines for the development of isoform specific cyclophilin ligands. A detailed comparison of the merits and drawbacks of the experimental and computational techniques used in this work has also been made, and strategies for effective combination of the methodologies in structure-based projects are outlined.
12
Thompson, Mary Katherine. „Functions of the eukaryote-specific ribosomal protein Asc1 /RACK1 in gene-specific translational activity“. Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/101355.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references.
Although the ribosome operates as a single molecular entity, it is composed of both ribosomal RNAs and dozens of proteins. However, the individual contributions of most ribosomal components to translational regulation are unknown. In Chapter 1, I will review the current state of knowledge related to the functions of ribosomal proteins with a focus on the RACK1 protein (Asc1 in yeast), a eukaryote-specific ribosomal protein with many proposed functions in both cellular signaling and translation. In Chapter 2, I will present evidence that the Asc1 protein is required for efficient translation of a specific set of mRNAs with short open reading frames (ORFs), including those that encode ribosomal proteins and nuclear-encoded mitochondrial components. Consistent with these translation defects, ASC1 mutants are unable to grow in conditions requiring full mitochondrial function. Asc1-sensitive mRNAs are highly associated with the translational closed-loop complex, a group of proteins that promotes a loop-like conformation of the mRNA during translation by simultaneous interaction with the 5' and 3' ends of the mRNA molecule. In wild type cells, mRNAs that associate strongly with the translational closed-loop complex are much shorter than other ORFs. Thus, I hypothesize that the closed-loop is preferentially formed and/or stabilized on mRNAs with short ORFs, and that this process is enhanced by the presence of Asc1 on the small ribosomal subunit. The dependence of closed-loop formation on ORF length could also explain why short ORFs have notably higher translation efficiency than longer ORFs, a trend I observed in data collected from several eukaryotes. In Chapter 3, I will present evidence that the mammalian RACK1 protein is also required for expression of mRNAs with short ORFs and for mitochondrial function in HeLa cells, similar to my observations in yeast. These findings hint at a conserved role for the Asc1/RACK1 protein in promoting the function of the closed-loop complex and the translation of short ORFs, which encode a set of highly abundant proteins required for central metabolic functions. Chapter 4 will discuss the biochemical and cell physiological implications of these findings and suggest some avenues for future research.
by Mary Katherine Thompson.
Ph. D.
13
Kimura, Tetsunari. „Contributions of specific intraprotein and protein-water interactions to the protein folding mechanism“. 京都大学 (Kyoto University), 2005. http://hdl.handle.net/2433/144930.
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14
Dahche, Hanan Mohamad. „Dual-specific protein phosphatases in the Archaea“. Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/37625.
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Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)C(X)₅R(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members.
ORFs encoding potential cPTPs have been identified in five archaeal species: Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Thermococcus kodakaraensis, Pyrococcus horikoshii, and S. solfataricus. Only one has been partially characterized, Tk-PTP from T. kodakaraensis. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented.
The genome of S. solfataricus encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T).
Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role.
ORF sso2453 from the genome of Sulfolobus solfataricus, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in E. coli and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in S. solfataricus cell lysates. Several potential substrates were isolated and identified by mass spectroscopy.Ph. D.
15
Gokoo, Suzanne. „Secretion of GBP, an infective stage-specific protein of Leishmania major“. Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265838.
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16
Chiu, Peng-hang Raymond, und 趙炳铿. „Identification of protein-interacting partners of testis-specific protein y-encoded like 2 (TSPYL2)“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41508725.
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17
Chiu, Peng-hang Raymond. „Identification of protein-interacting partners of testis-specific protein y-encoded like 2 (TSPYL2)“. Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508725.
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18
Lortie, Karine. „The growth-arrest-specific protein gas7 potentiates neuronal differentiation“. Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26701.
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The growth-arrest-specific gas7 protein is involved in neuronal development. Its role in neuronal differentiation and its potential neuroprotective activity were investigated in PC 12 and NT2 cells. gas7 overexpression in PC12 cells promoted neurite outgrowth and potentiated nerve growth factor-induced expression of the neuronal markers betaIII-tubulin, synaptotagmin, alpha7 subunit of the acethylcholine receptor, and dihydropyrimidinase related protein-3. This effect was exerted independently of cellular proliferation, as gas7 did not affect cell cycle progression. Endogenous gas7 expression was induced during neuronal differentiation of NT2 cells with retinoic acid, suggesting a role for gas7 in neuronal development. Finally, gas7 overexpression in PC 12 cells did not protect against toxicity triggered by oxygen-glucose deprivation, the calcium ionophore A23187 or sodium nitroprusside. The ability of gas7 to potentiate neuritogenesis and neuronal differentiation makes it a potential therapeutic target to promote re-establishment of neuronal connections in the injured or diseased brain, such as following stroke.
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Leddy, John J. „Molecular characterization of muscle-specific calmodulin-dependent protein kinases“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/NQ45180.pdf.
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20
O'Brien, Richard Mark. „Studies on the insulin receptor tyrosine-specific protein kinase“. Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252645.
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21
Waterhouse, Mark Peter. „Specific targeting of FcγRIIIa using artificial scaffold protein variants“. Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/20522/.
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Fcγ Receptors (FcγRs) are cell-surface receptors for IgG that are expressed on immune cells including macrophages, monocytes and natural killer (NK) cells. Upon binding to IgG-containing immune complexes, FcγRs trigger cell-mediated effector functions that lead to the clearance of pathogenic material and immune homeostasis. However, aberrant activation of these pathways can result in autoimmune susceptibility and, as such, FcγRs are implicated in the pathogenesis of several autoimmune disorders. For example, FcγRIIIa, expressed on macrophages and NK cells, has been functionally and genetically linked to susceptibility for rheumatoid arthritis and is a valid target for several FcγR-mediated autoimmune disorders. Despite this, FcγRIIIa is currently under-exploited due to a lack of FcγRIIIa-specific agents, which results from the expression of the near identical FcγRIIIb on neutrophils. However, several FcγRIIIa-specific Adhirons (an engineered protein scaffold) have recently been described, which inhibit IgG binding and effector functions (phagocytosis and cytokine release) of THP-1 cells. This thesis aimed to fully characterise and enhance the properties of one of these recently described Adhirons (known as AdG3) through site-directed mutagenesis (SDM); Molecular Dynamics (MD) simulations; and fusion to the IgG1- or IgG2-Fc. SDM identified several key binding residues in the AdG3 variable regions through screening of AdG3 mutants by surface plasmon resonance (SPR). SPR also identified three mutants with enhanced affinity for FcγRIIIa and showed that AdG3 possesses background binding to the highly homologous FcγRIIIbNA1, but not FcγRIIIbNA2, which was supported by MD simulations that revealed the exquisite mode of AdG3 specificity for these receptors, which was mediated through the Arg18Ser polymorphism in the AdG3 binding site. Fusion to the IgG-Fc was not detrimental to the FcγRIIIa-AdG3 interaction, and fusion to the IgG2-Fc was observed to potentially enhance the affinity and specificity of AdG3 for FcγRIIIa. These findings could subsequently be utilised for further improvement of AdG3 affinity and specificity, as well as other clinically relevant parameters such as serum half-life.
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Bullenkamp, Jessica Isabell. „Tumour-specific regulation of apoptin by protein kinase C“. Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/tumourspecific-regulation-of-apoptin-by-protein-kinase-c(2e7dd4a3-e361-4ad1-8cf3-92d36f43b58e).html.
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Apoptin, the VP3 protein derived from chicken anaemia virus (CAV), induces tumour cell-specific apoptosis and therefore represents a potential anti-cancer therapeutic agent of the future. In human tumour cells, but not in normal cells, Apoptin is phosphorylated and subsequently translocates to the nucleus, which are essential important steps for its cytotoxic activity. Recently, the β isozyme of protein kinase C (PKCβ) was identified as a kinase phosphorylating Apoptin in multiple myeloma cells. However, the exact mechanism and nature of interaction between PKCβ and Apoptin as well as its importance for Apoptin-induced cell death remain to be characterised. This project aimed to further investigate the physical and functional interaction between PKC isoforms, in particular PKCβ, and Apoptin. Recently, the first human Gyrovirus (HGyV) was isolated which shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin. Using a synthetic HGyV-Apoptin construct the subcellular distribution and apoptotic function of this novel Apoptin protein in several human cancer cell lines was analysed and compared to CAV-Apoptin. The results demonstrated a comparable tumour-specific nuclear translocation and cytotoxic effect between the two proteins. A model of colon carcinoma and normal mucosa cell lines was used to investigate the functional link between PKC and Apoptin in other cancer types. Immunoprecipitation and proximity ligation assay studies confirmed the binding of both CAV- and HGyV-Apoptin to PKCβI in HCT116 colorectal cancer cells and identified the N-terminal region of Apoptin to be important for the interaction with PKC. In contrast to HCT116 cells two normal colon mucosa cell lines tested expressed low levels of PKCβI. This differential expression pattern of PKCβI correlated with reduced Apoptin activation in normal cells, as evident by its localisation to the cytoplasm, decreased phosphorylation and lack of cytotoxic activity. Further studies revealed that PKCβI knockdown in HCT116 cells resulted in reduced Apoptin phosphorylation but did not impair Apoptin cytotoxicity. Additionally, overexpression of PKCβI was not sufficient to activate Apoptin in normal cell lines, indicating that other kinases or processes may contribute to the tumour-specific function of Apoptin. Using the FRET-based PKC activity reporter CKAR and fluorescence lifetime imaging microscopy (FLIM) the effect of Apoptin or other stimuli on PKC activity was analysed. Similar to treatment with the phorbol ester TPA, Apoptin expression in HCT116 cancer cells resulted in a significant increase in PKC activity, correlating with the expression and increased phosphorylation levels of Apoptin. In contrast, normal colon mucosa cell lines with low PKCβI expression showed a delayed and reduced induction of PKC activation in response to Apoptin. Overexpression and knockdown studies in combination with FLIM provided some evidence that Apoptin predominantly activates the PKCβI isoform in HCT116 cells. Another tumour-selective cytotoxic agent, TRAIL, was tested on a panel of head and neck cancer cell lines associated with human papillomavirus (HPV) infection. Addition of the proteasome inhibitor Bortezomib sensitised TRAIL-resistant HPV positive head and neck cancer cells to TRAIL. This seemed to involve activation of caspases, the anti-apoptotic protein XIAP as well as stabilisation of functional p53, but the precise mechanism has so far not yet been established. In conclusion, the results of this study propose an important link between Apoptin and PKCβI in cancer cells, involving their interaction and co-localisation, Apoptin-induced activation of PKC and PKCβI-mediated phosphorylation of Apoptin to promote its nuclear translocation and cytotoxic function. In addition, the novel HGyV-Apoptin was shown to function in a similar manner to CAV-Apoptin, providing a further tumourspecific protein for future studies.
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Cucchetti, Margot. „Rltpr, a lymphoid-specific protein essential for CD28 costimulation“. Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4043.
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La reconnaissance d'antigènes par le TCR active des protéines tyrosine kinases qui phosphorylent d'autres substrats intracellulaires dont LAT. Ceci engendre l'activation de molécules telles que PKCθ et CARMA-1. La mutation LatY136F associe des TCR "estropiés" dans le développement de cellules T effectrices générant des désordres lymphoprolifératifs. Nous avons essayé de comprendre les gènes aggravant ou empêchant cette lymphoprolifération en utilisant la mutagénèse ENU. Nous avons identifié une mutation appelée Basilic empêchant le déroulement de la pathologie LatY136F. Basilic est une mutation du gène Rltpr qui constitue une phénocopie de Cd28-/- sur fond sauvage et sur fond LatY136F. Rltpr est un une nouvelle protéine ayant de multiples domaines, qui appartient à la famille CARMIL et qui est exprimée dans les cellules T et B. L'objectif de ce travail était d'élucider les mécanismes au cours desquels CD28 et Rltpr coopèrent avec le TCR pour différencier des cellules T naïves en cellules T effectrices. Ce travail visait aussi à caractériser Rltpr, dont la structure/fonction et l'interactome sont encore inconnus. En utilisant des techniques de microscopie confocale, nous avons montré que la localisation et le recrutement de Rltpr et de RltprBas à la synapse immunologique sont tous deux CD28-dépendants. Les deux molécules colocalisent avec CD28 tout au long du processus d'activation. En outre, Rltpr est essentiel pour la translocation à la synapse de PKCθ et CARMA-1, qui sont induits lors de la co-stimulation par CD28. Ces résultats permettent une meilleure compréhension du fin réglage du système immunitaire adaptatif qui est mis en place lors de l'activationTCR recognition of antigens triggers the activation of protein tyrosine kinases that phosphorylate other intracellular substrates including LAT. LAT phosphorylation leads to the activation of PKCθ and CARMA-1. The point mutation LatY136F associates TCRs with crippled signaling abilities to the development of effector T cells generating lymphoproliferative disorders (LPDs). We tried to shed light on genes exacerbating or preventing the LatY136F LPD by using an ENU mutagenesis screening. We identified one point mutation called Basilic that prevents the unfolding of the LatY136F pathology. Basilic is a point mutation of the Rltpr gene and is a phenocopy of a Cd28-/- mutation both on a wild-type and on a LatY136F background. Rltpr is a newly-discovered, multidomain protein belonging to the CARMIL family that is expressed in T and B cells. The objective of the present work was to elucidate the mechanisms during which CD28 and Rltpr cooperate withthe TCR to differentiate naïve into effector T cells. I also aimed at characterizing the Rltpr molecule, whosestructure/function and interactome are still largely unknown. Using confocal microscopy in collaborationwith Takashi Saito's group and Christoph Wülfing we showed that the localization and the recruitment ofboth Rltpr and RltprBas at the immune synapse are CD28-dependent. The two molecules colocalize with CD28all along the activation process. Moreover, Rltpr is essential for the synapse translocation of PKCθ andCARMA-1, which are induced upon CD28 costimulation. Those results allow a better understanding of theadaptive immune system fine tuning upon activation
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Ooi, Aik Teong. „Sequence-Specific DNA Detection Utilizing Custom-Designed Zinc Finger Proteins“. Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/194236.
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DNA diagnostics are important technologies in molecular and cellular biology. By allowing identification of specific sequences, DNA-based diagnostics potentially provide more accurate and rapid results than protein- or antigen-based diagnostics, primarily because phenotypic changes come much later than changes in genotype. Despite this advantage, there are fewer diagnostic or imaging systems that target DNA than those targeting proteins, antibodies, or antigens.Each type of DNA-based diagnostic has its own, unique set of limitations; however, most can be attributed to issues related to sequence restriction, signal detection, specificity, or some combination thereof. For example, while PCR-based methods allow amplification and assessment of specific DNA sequences, they lack the ability to report information of specific cells, or cell types, within the heterogeneous pool of cells typically found in a tumor biopsy. In addition, none of the currently available DNA detection methods has the potential to be utilized in living cells, a disadvantage which limits the potential applications.The work presented here describes the design and development of a new methodology for the detection of specific double-stranded DNA sequences. This detection method is based on the concept that two inactive fragments of a reporter protein, each coupled to engineered zinc finger DNA-binding motifs, are able to reassemble and form an active complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), can achieve single base-pair specificity, and has the potential to be utilized in living cells.In this dissertation, we discuss the efforts from constructing to refining the system, as well as the future applications of SEER in diagnostics and therapeutics. Chapter I will provide an introduction to DNA detection methods, on which the principles of the SEER system are based. Chapter II describes the design and construction of an enzymatic SEER system, SEER-LAC, using beta-lactamase as the enzyme. In Chapter III, we outline the in vitro characterization of the SEER-LAC system, followed by its optimization in Chapter IV. Chapter V illustrates the efforts to develop SEER system for mammalian cell culture applications. In the final chapter, the future developments and applications of SEER are discussed.
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Feng, Bochen. „Specific DNA-Protein and Protein-Protein interactions determine the operation of the Nitrogen regulatory circuit of Neurospora Crassa /“. The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488192447431226.
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Yip, Kit-yan. „Identification of the protein interacting partners of testis specific protein, Y-encoded like-2 (TSPYL2)“. Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557844.
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Yip, Kit-yan, und 葉潔茵. „Identification of the protein interacting partners of testis specific protein, Y-encoded like-2 (TSPYL2)“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557844.
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Jeong, Dae Gwin. „Crystal structures of dual specific protein phosphatases and insight into function of protein tyrosine phosphatase“. 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/136974.
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Wang, Yanchao. „Protein Structure Data Management System“. Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/cs_diss/20.
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With advancement in the development of the new laboratory instruments and experimental techniques, the protein data has an explosive increasing rate. Therefore how to efficiently store, retrieve and modify protein data is becoming a challenging issue that most biological scientists have to face and solve. Traditional data models such as relational database lack of support for complex data types, which is a big issue for protein data application. Hence many scientists switch to the object-oriented databases since object-oriented nature of life science data perfectly matches the architecture of object-oriented databases, but there are still a lot of problems that need to be solved in order to apply OODB methodologies to manage protein data. One major problem is that the general-purpose OODBs do not have any built-in data types for biological research and built-in biological domain-specific functional operations. In this dissertation, we present an application system with built-in data types and built-in biological domain-specific functional operations that extends the Object-Oriented Database (OODB) system by adding domain-specific additional layers Protein-QL, Protein Algebra Architecture and Protein-OODB above OODB to manage protein structure data. This system is composed of three parts: 1) Client API to provide easy usage for different users. 2) Middleware including Protein-QL, Protein Algebra Architecture and Protein-OODB is designed to implement protein domain specific query language and optimize the complex queries, also it capsulates the details of the implementation such that users can easily understand and master Protein-QL. 3) Data Storage is used to store our protein data. This system is for protein domain, but it can be easily extended into other biological domains to build a bio-OODBMS. In this system, protein, primary, secondary, and tertiary structures are defined as internal data types to simplify the queries in Protein-QL such that the domain scientists can easily master the query language and formulate data requests, and EyeDB is used as the underlying OODB to communicate with Protein-OODB. In addition, protein data is usually stored as PDB format and PDB format is old, ambiguous, and inadequate, therefore, PDB data curation will be discussed in detail in the dissertation.
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Kepple, Kevin V. „Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro /“. Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208620.
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Pelletier, Marie-Eve. „PRMT8: Characterization of a novel neuron-specific protein arginine methyltransferase“. Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28467.
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Methylation of arginine residues is a post-translational modification mediated by specific enzymes known as the Protein Arginine Methyltransferase (PRMT) family. In this thesis, we present our research on PRMT8, an enzyme catalyzing the formation of asymmetric dimethylarginine (aDMA), which displays a unique distribution at the plasma membrane of neuronal cells of the central nervous system (CNS). An ontogenic analysis of PRMT8 in mouse tissues revealed that its expression in the brain is induced during the perinatal stage and is maintained in adulthood. The P19 cell line was identified as a valid model for endogenous PRMT8 and showed the induction and requirement of PRMT8 to achieve neuronal processes. A P19 PRMT8 knock-down cell line does not survive neuronal differentiation while, in contrast, no cell death is observed when the muscle lineage is induced. Taken together, these findings suggest an important role for PRMT8 in neuronal differentiation and/or function in the CNS.
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Edwards, Malia Michelle 1975. „Alteration of astrocyte-specific protein expression : implications for Alzheimer's disease“. Monash University, Dept. of Psychology, 2002. http://arrow.monash.edu.au/hdl/1959.1/7859.
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Baker, Stacy. „Structural characterization and ligand specific protein interactions of androgen receptor“. Connect to resource, 2008. http://hdl.handle.net/1811/31785.
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Alce, Timothy Mark. „Characterisation of a novel stage-specific protein from Leishmania major“. Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285161.
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Chan, Tung-lei, und 陳冬妮. „Promoter characterization of testis specific protein, Y-linked like2 (TSPYL2)“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290410.
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Hardie, Sharon Shillinglaw. „Nucleotide analogues as reagents for site-specific protein-DNA crosslinking“. Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394811.
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Balan, Sibu. „Disulfide bridging-poly (ethylene glycol) reagentsfor site-specific protein conjugation“. Thesis, University College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444180.
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Popp, Maximilian Wei-Lin. „Site-specific protein labeling via sortase A and its applications/“. Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/65294.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references.
Technological improvements in the assays and equipment used for biological, biochemical, biophysical and microscopy purposes have ensured that methods for labeling of proteins with reporter molecules remain in high demand. Standard chemical labeling methods using entities that react with amino acid side chains lack the specificity to ensure precise placement of reporter groups. Genetic methods, although specific, lack the versatility afforded by chemical synthesis-most reporters are limited to protein sized domains or peptide tags to which corresponding antibody based reagents are available. The first portion of this work is devoted to the establishment of a system that allows for the site-specific labeling of proteins with a wide variety of chemically synthesized probes. This system exploits sortases, a class of bacterial transpeptidases, that recognize a small five amino acid tag genetically fused to the protein of interest and catalyze the formation of an amide bond between the protein to be studied and the probe. The second part of this thesis describes how this sortase mediated protein labeling method has been implemented to explore enzyme structure and function, improve the physical properties of therapeutic proteins, study glycoproteins important for innate immune responses in living cells (Appendix A), and visualize influenza glycoproteins in living, infected cells. Finally, a protocol is included for this system (Appendix B), which is both versatile and easy to establish in any lab. The synthetic chemistry demanded is minimal, requiring only standard, readily available reagents, making the system amenable to labs equipped for cell and molecular biology experiments.
by Maximilian Wei-Lin Popp.
Ph.D.
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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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Kwon, Inchan Tirrell David A. „Protein engineering via site-specific incorporation of nonnatural amino acids /“. Diss., Pasadena, Calif. : Caltech, 2007. http://resolver.caltech.edu/CaltechETD:etd-01222007-010333.
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Fan, Jun. „The functional studies of a novel Golgi specific spectrin protein /“. For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2003. http://uclibs.org/PID/11984.
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Cardinale, Stefano. „Inactivation of a human kinetochore by specific targeting of chromatin modifiers“. Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/3815.
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Here I describe the construction and characterization of a new generation of human artificial chromosome that contains an array of DNA sequences that can be used to manipulate the chromosome in vivo and possibly in vitro. This HAC was originated in human fibrosarcoma HT1080 cells from a synthetic alphoid DNA containing an array of TetOperator sequences, cloned in a BAC-based vector. This synthetic ά-satellite DNA formed HACs that were stably maintained throughtout replication and segregation in HT1080 cells. However, I succeeded to also transfer and manipulate the alphoidtetO HAC into a HeLa-based hybrid cell line. The synthetic alphoidtetO HAC chromatin was similar to the chromatin at endogenous centromeric alphoid DNA. Importantly, the DNA sequences embedded in the synthetic HAC were accessible to targeting TetR-fused constructs in vivo. The alphoidtetO HAC could be successfully targeted with a number of TetR:fusion proteins without affecting its chromatin structure, kinetochore assembly and mitotic behaviour. However, the targeting of a transcriptional activator (tTA) inactivated the HAC synthetic alphoidtetO DNA in a fraction of transfected cells. Surprisingly, the targeting of the transcriptional repressor tTS, co-repressor KAP1 or the heterochromatin-associated protein HPIά severely inactivated the synthetic alphoidtetO kinetochore . In fact, upon targeting several inner and outer kinetochore proteins were delocalized from the alphoidtetO sequences. The dissociation of kinetochore proteins CENP-H and CENP-C appeared to precede that of CENP-A. The alphoidtetO HAC lacking inner kinetochore protein complexes showed mitotic defects including misalignment at the metaphase plate and defective anaphase segregation, ultimately being included in tiny DAPI-positive nano-nuclei in the cytoplasm. The transcriptional repressor tTS repressed the low levels of transcription from the alphoidtetO sequences. In addition, targeting of transcriptional repressors altered the HAC chromatin towards a more “closed”, heterochromatic conformation, as seen from the changes in histone tail modifications. Interestingly, the targeting of the histone methyltransferase EZH2 to the alphoidteto HAC showed a much milder inactivating activity compared to KAP1. Based on these results, I propose that the formation of HPI-type of heterochromatin or accumulation of HPIά to the centromeric regions could disrupt the association of constitutive kinetochore proteins to the underlying sequences. Centromeric alphoid sequences lacking a functional kinetochore structure then also loose the centromere-specific histone H3 variant CENP-A becoming definitively inactive. Alternatively, a basal transcriptional activity from centromeric sequences might be required for centromere functionality.
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Sizer, P. J. H. „Studies of specific molecular interactions within and between membrane bilayers“. Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233640.
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Thorpe, Helena M. „Site-specific recombination in Streptomyces temperate phage #pi#C31“. Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285769.
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Lyons, James Geoffrey. „Enhanced Feature Extraction from Evolutionary Profiles for Protein Fold Recognition“. Thesis, Griffith University, 2016. http://hdl.handle.net/10072/365732.
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Proteins are important biological macromolecules that play important roles in al- most all biological reactions. The function of a protein is dependent on the shape it folds in to, which is in turn dependent on the protein’s amino acid sequence. Ex- perimental approaches for determining a protein’s 3D structure are expensive and time consuming, so computational methods for determining the structure from the amino acid sequence are desired. Methods for directly computing the 3D structure of a protein exist, however they are impractical for large proteins and high resolution models due to the large search space. Instead of trying to directly find the 3D struc- ture from first principles, the primary structure can be compared to proteins with known 3D structure. A ‘fold’ is a way of classifying proteins with the same major secondary structures in the same arrangement and with the same topological con- nections. Protein Fold Recognition (PFR) is an important step towards determining a protein’s structure, simplifying the protein structure prediction problem. This is a multi-class classification problem solvable using machine learning techniques.
The PFR problem has been widely studied in the past, with feature extraction approaches including using counts of amino acids and pairs of amino acids, physic- ochemical information, evolutionary information from the Position Specific Scoring Matrix (PSSM), and structural information from its predicted secondary structure. These approaches do work, but with limited success. Current state of the art features use information from the PSSM as well as the predicted secondary structure.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith School of Engineering
Science, Environment, Engineering and Technology
Full Text
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Willis, Brandon S. „Cell-type specific activation of a Protein Kinase A inhibitory mutation in mice /“. Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5053.
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Rangarajan, Desikan. „Characterisation of an infective-stage specific surface protein of Leishmania major“. Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338875.
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Trowern, Angus Robert. „Regulation of the neurone-specific protein gene product (PGP) 9.5 gene“. Thesis, University of Southampton, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262908.
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Lenoir-Capello, Rachel. „Specific labeling strategies for new developments in liquid state protein NMR“. Thesis, Sorbonne université, 2020. https://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2020SORUS056.pdf.
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La résonance magnétique nucléaire (RMN) fournit des informations structurelles et dynamiques précieuses à l'échelle atomique, cependant, la faible sensibilité et résolution des signaux empêchent l’étude d'objets moléculaires plus importants. Nous présentons 3 stratégies de marquage isotopique pour différentes expériences RMN des protéines en solution et démontrons leur potentiel pour l'étude structurale des biomolécules. Parmi les stratégies envisagées, 2 utilisent l'expression in vitro pour obtenir des protéines marquées sélectivement sur un groupe chimique et/ou acide aminé dans un environnement perdeutéré. Avec l’utilisation de séquences d'impulsions TROSY, ces échantillons ont permis des gains spectraux importants lorsque ils étaient spécifiquement marqués sur des groupes amide ou sur le méthylène des glycines tout en maintenant un taux de deutération élevé sur les autres fonctions chimiques des protéines. La troisième stratégie de marquage protéique utilise des protocoles in vivo pour des applications RMN innovantes: l'hyperpolarisation de noyaux en solution qui augmente leur sensibilité de plusieurs ordres de grandeur. La durée de vie de cette hyperpolarisation est régie par le temps de relaxation longitudinale des noyaux, qui est réduit pour les protéines à température ambiante. En isolant les noyaux d'intérêt dans un environnement perdeutéré, les interactions dipolaires créées par les protons voisins sont éliminées et les noyaux hyperpolarisés relaxent beaucoup plus lentement. L'hyperpolarisation d'un petit domaine protéique a été entreprise avec succès mais les conditions de dissolution doivent encore être améliorées pour conserver une phase aqueuse homogèneNuclear Magnetic Resonance (NMR) provides valuable structural and dynamic information at the atomic scale, however, the low sensitivity and resolution of signals rapidly preclude investigations of larger molecular objects. We present three isotopic labeling strategies for different protein-solution NMR experiments and demonstrate their potential for the structural study of biomolecules in solution. Among the strategies considered, two are based on the use of in vitro protein expression to obtain selectively labeled proteins of a certain chemical group and/or amino acid in a perdeuterated environment. Perdeuteration is essential for the optimal use of Transverse Relaxation Optimized Spectroscopy pulse sequences. They allowed significant spectral gains when samples were specifically labeled on amide groups or on the methylene of glycines while maintaining a very high rate of deuteration on the other chemical functions of the proteins. The third protein labeling strategy employed is based on in vivo protocols but used in innovative NMR applications: a technique of hyperpolarization of nuclei in solution which increases their sensitivity by several orders of magnitude. The lifetime of this hyperpolarization is governed by the longitudinal relaxation time of nuclei, which are reduced for proteins at room temperature. By isolating the nuclei of interest in a perdeuterated environment, dipolar interactions created by neighboring protons were eliminated and hyperpolarized nuclei relaxed much more slowly. Hyperpolarization of a small protein domain was successfully undertaken at 1K but the dissolution conditions need to be improved in order to preserve a homogeneous aqueous phase
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Chan, Tung-lei. „Promoter characterization of testis specific protein, Y-linked like 2 (TSPYL2)“. Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290410.
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