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1
Boyd-Kirkup, Jerome Douglas. "Characterising putative mammalian mitochondrial nucleoid proteins". Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608921.
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Cao, Wei, i 曹威. "Structural studies of two nucleoid-associated proteins : histone-like nucleoid-structuring protein H-NS and α-hemolysin expression-modulating protein Hha". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208420.
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In prokaryotic cells, the nucleoid contains almost all the genetic materials as well as a number of nucleoid structuring factors. The nucleoid-associated proteins (NAPs) are known to have low molecular weight and the ability to form dimer or oligomer, and most of them can bind to DNA for regulation of gene expression. The Histone-like nucleoid structuring protein H-NS, well studied as one of the NAPs, acts as a global transcriptional repressor. It has independent functional N-terminal domain for oligomerization and C-terminal domain for DNA binding, joined by a flexible linker. H-NS contributes to horizontal genes transfer and responses to environmental factors like temperature or pH, which would influence the oligomerization ability of H-NS and DNA binding. The α-hemolysin expression-modulating protein Hha is a member of the Hha-YmoA family, expressed only in Gram-negative Enterobacteriaceae as a modulator of virulence factors expression. In E. coli, the binding of Hha to H-NS can modulate the expression of α-hemolysin operon, which is essential for the H-NS-regulated gene expression. In this study, both Hha and the oligomerization domain of H-NS (H-NS64) were expressed in E. coli and the purified proteins were crystallized. The Hha crystals diffracted to 2.2 Å; and the HhA/H-NS complex crystals diffracted to 1.8 Å. Both structures were successfully determined by molecular replacement method. Comparisons were carried out between the published apo Hha and H-NS structures and our complex structures. The structures showed the binding details between H-NS and Hha and also conformational changes of each protein, which may indicate how Hha regulates gene expressions through H-NS.published_or_final_version
Physiology
Master
Master of Philosophy
3
Bradshaw, Elizabeth Helen. "Nucleoid-associated proteins of Streptomyces coelicolor : discovery and functions". Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/44850/.
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The regulation of gene expression from biosynthetic gene clusters is a key concern in natural product discovery as these clusters are often transcriptionally silent (“cryptic”) under normal laboratory conditions, making the initial characterisation and heterologous expression of the products they encode problematic. The role of the architectural nucleoid-associated proteins (NAPs) in regulation of the expression of these products has been neglected within the Actinomycetes. NAPs are small, highly abundant proteins which govern both gene expression and nucleoid structure on a genome-wide scale. A method for surveying the proteome of the S. coelicolor nucleoid was developed which generated a list of 25 proteins with a high probability of being NAPs. This list included known NAPs such as HupA, HupS, Lsr2 and sIHF and the known global regulators CRP and BldD, as well as a number of interesting novel NAP candidates from a variety of protein classes suitable for further investigation. One of these proteins, SCO5592, was investigated as a candidate global RNA-binding regulator. It was found to comprise a KH domain with an N-terminal extension and formed oligomers of 10 - 12 subunits in solution. The mutant phenotypes of both S. coelicolor paralogs of HU (HupA and HupS) were examined and showed opposite effects on growth, spore formation and actinorhodin production. The mutant phenotypes of both S. coelicolor paralogs of the H-NS-like proteins Lsr2 (SCO3375 and SCO4076) were milder and showed a greater degree of overlap than the HU mutants, however both showed a moderate abnormality in spore morphology, suggesting that they have a role in spore nucleoid segregation. Phylogenetic analysis of the HU and Lsr2 proteins revealed that the lysine-rich tail domain of HupS was acquired after the HupA and HupS core domains had begun to diverge and that lysine-rich sequences have evolved multiple times.
4
Ono, Shusuke. "The biophysical characterisation of the Enterobacterial nucleoid proteins H-NS and StpA". Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1445757/.
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H-NS is a major protein component of the nucleoid, found in many Gram-negative bacterial species. H-NS is involved in the modulation of expression of a wide range of genes, as well as contributing towards nucleoid structure. There are two structur ally independent domains in H-NS, one involved in protein-protein interactions (the N-terminal oligomerisation domain), and the other involved in DNA-binding (C- terminal). These are joined via a flexible linker sequence. H-NS both self-associates and interacts with other nucleoid-associated proteins to form specific oligomeric complexes that bind DNA, allowing a precise level of control in gene expression. The protein StpA, a paralogue of H-NS with 58% sequence identity, was originally identified by its RNA chaperone activity. Subsequent studies have suggested struc tural similarities between H-NS and StpA, with a degree of overlap in their function in vivo. StpA self-associates in a similar manner to H-NS, and has been shown to in teract with H-NS. Whilst small differences in the properties of H-NS and StpA have been identified, no clear distinctions have been made between the two proteins. This work investigates several key issues regarding the properties of the StpA protein. A number of biophysical techniques have been used to investigate the interaction of H-NS and StpA. The results of these experiments are consistent with a model whereby StpA self-associates via a 'head-to-taif interaction. Furthermore, StpA ex hibits a different affinity and kinetic behaviour of association in comparison to H-NS. The properties of self-association and interaction of H-NS and StpA are fully consis tent with studies that highlight the intimate relationship between the two proteins in vivo. Two independent structures of the N-terminal oligomerisation domain of H-NS have been reported. These structures were derived at different temperatures (i.e. 25 C and 35 C). To investigate the implications of these model structures on the thermoregula tory functions of H-NS, the oligomerisation properties of H-NS were investigated over a temperature range. Both the oligomerisation and DNA-binding properties of H-NS were found to vary within a physiologically relevant range of temperatures. To characterise the interaction of StpA with DNA and to allow comparison with H- NS, the solution structure of the C-terminal domain of StpA was determined, solved by NMR. The interaction of DNA with this domain was characterised using both NMR and calorimetric methods. Statement The work described in this thesis was carried out in the Department of Biochemistry and Molecular Biology, University College London between 2000 and 2004. The NMR spectra were acquired with the assistance of Dr. M. Williams, Dr. R. Harris or J. Taylor. All experiments involving H-NS 1.39 were conducted with the assistance of T. Olsson. All other work was carried out by the author. This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC). Some of the work detailed in this thesis has been published elsewhere: Esposito, D., Petrovic, A., Harris, R., Ono, S., Eccleston, J. F., Mbabaali, A., Haq, I., Higgins, C. F., Hinton, J. C, Driscoll, P. C, and Ladbury, J. E. (2002) H-NS oli gomerisation domain structure reveals the mechanism for high order self-association of the intact protein. J Mol. Biol. 324, 841-850.
5
Mahajan, Shikha. "Protein Profiling of Adenine Nucleoside and Nucleotide Analogs Binding Proteins Using N6-Biotinylated-8-azidoadenosine Analogs as Affinity Based Protein Profiling Probes". Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4139.
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Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest.
Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over
nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins.
To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.
6
Johansson, Monika. "The role of nucleoside diphosphate kinase in plant mitochondria /". Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200674.pdf.
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Wallis, Anne Elizabeth. "Identification of Leishmania genes encoding proteins containing tandemly repeating peptides". Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/29447.
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In order to identify Leishmania proteins which may be immunologically relevant or may play a role in interactions between Leishmania and its mammalian host, a Leishmania major genomic DNA library was constructed in the vector λgt11 and screened with antibodies raised to Leishmania major promastigote membranes. Two recombinant DNA clones were identified which encoded repetitive sequences (Clone 20 and Clone 39). Clone 20 encoded a repetitive peptide of 14 amino acids and clone 39 encoded an unrelated repetitive peptide of 10 amino acids. Analysis of one of these clones, Clone 20, indicated that there were two RNA transcripts of 9500 and 5200 nucleotides expressed which corresponded to this clone in Leishmania major and Leishmania donovani and this expression was not stage-specific. The results of genomic DNA analysis and isolation of additional clones encoding Clone 20 sequences indicated that there were two genes which corresponded to Clone 20 in both Leishmania major and Leishmania donovani and that these genes differed from one another with respect to the number of repeats which they contained. Antibodies against the fusion protein produced by Clone 20 recognized a series of Leishmania major proteins of apparent mol wt 250,000. Analysis of Clone 39 indicated that there was a single transcript of 7500 nucleotides expressed which corresponded to this clone in both Leishmania major and Leishmania donovani and that there was a single gene (or two identical genes) which encoded this transcript.
The genomes of many protozoan parasites exhibit a high degree of plasticity with respect to chromosome size and number. The presence of highly repetitive regions within their DNA may be involved in maintaining this plasticity, allowing the parasite to evolve rapidly under selective pressure. Repetitive regions have been identified within many Plasmodia antigens and have been implicated in the ability of this parasite to evade the host immune system. The presence of Leishmania genes encoding proteins containing tandemly repeating peptides may indicate that these proteins play a similar role in evading the host immune system during the course of Leishmania infections. The possible evolution and functions of repetitive proteins in protozoan parasites is discussed.Medicine, Faculty of
Medical Genetics, Department of
Graduate
8
Hamilton, Tatyana. "Protein-nucleic acid interactions of Wilms' tumor and TFIIIA zinc finger proteins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34266.pdf.
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Bramble, Sharyl Elizabeth. "Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium discoideum". Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26172.
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One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides
like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography.
A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence
of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides
such that GTP binding was not detectable in these experiments
or that the ras protein from D. discoideum simply does not bind guanine nucleotides.
The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched
relative to other proteins because the immunoaffinity columns did not bind p23RAS.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
10
Pérez, González Daniel Cibrán. "Single-molecule studies of nucleic acid folding and nucleic acid-protein interactions". Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/12039.
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Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.
11
Nadassy, Katalin. "Molecular recognition by proteins : structural features of zinc and protein-nucleic acid binding sites". Thesis, University of Stirling, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341227.
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Cirillo, Davide. "Prediction of protein and nucleic acid interactions". Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/403537.
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The purpose of my doctoral studies has been the development of bioinformatics methods to quantitatively evaluate associations between proteins and nucleic acids (NAs). This thesis aims at providing insights into molecular features and still relatively unknown mechanisms of protein-NAs associations, such as RNA-binding proteins and long noncoding RNAs as well as transcription factors and regulatory DNA elements. In this work, I present two algorithms, catRAPID omics express and PAnDA, for the prediction of RNA- and DNA-protein interaction respectively. Those computational methods offer the possibility to address experimental problems and guide new approaches largely facilitating experimental design and proceduresMis estudios de doctorado han tenido como propósito principal el desarrollo de herramientas bioinformáticas para la evaluación de interacciones entre proteínas y ácidos nucleicos (ANs) de forma cuantitativa. Por consiguiente, esta tesis apunta a proporcionar conocimientos sobre características moleculares y mecanismos de asociación proteína-AN aún relativamente desconocidos; concretamente, la asociación de proteínas a ARNs y ARNs no codificantes, a la vez que factores de transcripción y elementos de regulación del ADN. En este proyecto presento dos algoritmos: catRAPIDomics express y PAnDA, cuyas finalidades son las de predecir interacciones proteína-ARN y proteína-ADN respectivamente. Dichos métodos computacionales ofrecen la posibilidad de abordar problemas experimentales, así como de guiar el diseño y procedimiento de nuevas estrategias para su resolución.
13
Furman, Jennifer Lynn. "IN VITRO AND IN VIVO DETECTION OF NUCLEIC ACIDS AND PROTEINS USING SPLIT-PROTEIN REASSEMBLY". Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/195828.
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The ability to directly monitor the presence of specific proteins or nucleic acids in a variety of in vitro and in vivo contexts has great utility for understanding biology as well as for the development of diagnostic agents. Herein I describe several methodologies, utilizing split-protein reassembly, which provides potentially general strategies for sequence-specific detection of DNA and RNA sequences as well as poly(ADP-ribose). I also provide a new split-protein approach for the direct detection of native proteins, such as the cancer marker HER2.The green fluorescent protein (GFP) provides a convenient sensor for reporting on a variety of cellular events. A series of spectroscopically distinct GFP variants was developed for sequence-specifically reporting on DNA. Each of these variants was demonstrated to provide a sensitive readout for the presence of a particular DNA sequence. Furthermore, utilizing a method of mixed split-protein complementation, I was able to simultaneously report on the presence of two distinct DNA sequences in the same solution.To provide a general solution for reporting on the presence of particular RNA sequences, a method was developed that utilized elements from a hybridization-based detection strategy coupled with split-protein reassembly. Specifically, DNA guide sequences complementary to an intended target were attached to hairpin sites that served as binding sites for high-affinity zinc fingers. Localization of the zinc fingers allowed for reassembly of the attached split enzyme, providing a sensitive readout for the presence of potentially any RNA sequence of interest. This methodology was applied to the detection of mRNA encoding VEGF, hDM2, and HER2, each of which may be overexpressed in cancer.A method was established for reporting on the presence of modifications to DNA and proteins that are elicited in response to DNA damage. Specifically, sensors were designed, which incorporated endogenous damage-recognition domains, to report on the global presence of particular DNA modifications, including the formation of 8-oxoguanine and pyrimidine dimers. Furthermore, to provide a technique for monitoring the general accrual of DNA damage and to interrogate the DNA damage response in cells, a sensor was developed which reported on the accrual of a posttranslational protein modification, poly(ADP-ribosyl)ation.Finally, I describe advances toward the adaptation of our protein-based biosensors for use in living cells, utilizing both GFP-based approaches for live cell imaging as well as luminescent-based strategies for reporting on proteins and nucleic acids following cell lysis.
14
Tisi, Dominic John Guiseppe. "Structural studies on nucleotide binding proteins". Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391822.
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Krasova, Alina [Verfasser]. "Identifying nucleoside- and nucleotide-protein interactions with S-adenosyl-L-homocysteine and adenosine triphosphate capture compounds / Alina Krasova". Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2013. http://d-nb.info/1031115560/34.
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16
Kimura, Koji. "Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction". Kyoto University, 2004. http://hdl.handle.net/2433/145287.
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Adhikari, Anirban. "Regulation of guanine nucelotide exchange in inhibitory G protein alpha subunit by activator of G protein signaling 3 and novel regulatory peptides". Embargoed access until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=114.
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Kneissl, Sabine. "Photocontrol of protein-protein and protein-nucleic acid interactions". Thesis, Cardiff University, 2009. http://orca.cf.ac.uk/54835/.
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Proteins often depend on a-helices for binding to other biomacromolecules. Reversible control of a-helix stability was accomplished in previous studies by incorporating a photoisomerisable azobenzene cross-linker into peptides, subsequently enabling the optical control of DNA-protein interactions. This approach was extended in this study to include protein-protein and protein-RNA interactions. One of the primary regulatory components in apoptosis signalling is the antiapoptotic protein Bcl-xL which interacts with the a-helical BH3 domain of the Bak protein. The Rev/RRE interaction is crucially involved in the life cycle of Human Immunodeficiency Virus. These interactions were targeted by designing peptides based on the BH3 domain of Bak and on the RNA-binding domain of Rev these peptides are activated by external light pulses after the incorporation of the cross-linker. The ability to control cross-linker conformation and hence peptide secondary structure was demonstrated by CD and UV/Vis spectroscopy. The binding to the target structure and complex disruption was determined in the dark-adapted and irradiated states using fluorescence based assays. Structural studies using NMR spectroscopy demonstrated that the alkylated peptides bind to the same part of the target molecule as the wild-type peptide, regardless of their structure. Moreover, one of the BH3 domain-based peptides and the light-controllable transcription factor PhotoMyoD were modified with protein transduction domains to enable future in vivo studies. Overall, this work opens the possibility to interfere reversibly and specifically with protein-protein and protein-RNA interactions and to study and modulate cellular function by optical control.
19
Gowda, Naveen Kumar Chandappa. "Regulation of Hsp70 function by nucleotide-exchange factors". Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-129118.
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Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
20
Clyde-Smith, Jodi. "Characterization of ras isoform activation by ras guanine nucleotide exchange factors /". St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16393.pdf.
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21
Mutomba, Martha Chengetai. "Guanine nucleotide-binding proteins of Trypanosoma brucei". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308280.
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22
McPherson, Cameron Scott. "Targeted knockdown of CREB1 in brain nuclei critically involved in drug-seeking behaviour /". Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/3518.
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23
Johansson, Kenth. "Structural studies of four nucleotide binding proteins : aldehyde dehydrogenase, NADP-malate dehydrogenase and two deoxynucleoside kinases /". Uppsala : Swedish University of Agricultural Sciences, 2000. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009416200&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Murakami, Shinya. "Biochemical characterization of CND41,a nucleoid DNA binding protein in chloroplast". Kyoto University, 2000. http://hdl.handle.net/2433/78113.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第8529号
農博第1138号
新制||農||807(附属図書館)
学位論文||H12||N3432(農学部図書室)
UT51-2000-J38
京都大学大学院農学研究科農芸化学専攻
(主査)教授 佐藤 文彦, 教授 關谷 次郎, 教授 大山 莞爾
学位規則第4条第1項該当
25
Bennett, Matthew Stuart. "Crystallography of biomolecular complexes, revealing protein-nucleoside, protein-protein acid-drug interactions". Thesis, King's College London (University of London), 2002. https://kclpure.kcl.ac.uk/portal/en/theses/crystallography-of-biomolecular-complexes-revealing-proteinnucleoside-proteinprotein-aciddrug-interactions(4a85b5cd-8a9a-4969-bee3-95fbe46e4257).html.
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Iuga, Adriana. "Solid-state 31P NMR of nucleotide binding proteins". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973225238.
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Heurtel, Thuswaldner Sophie. "Nucleotide-binding Proteins in the Plant Thylakoid Membrane". Licentiate thesis, Linköping Department of Biomedicine and Surgery, Linköping University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7934.
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Worth, Graham Alan. "The energetics of nucleotide binding to RAS proteins". Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:44524415-2f2b-4601-998c-56110f332153.
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Ras proteins are a special class of proteins that mediate cell growth signals. Their importance lies in the fact that they are products of a proto-oncogene. This means that under certain conditions the gene that determines its structure is altered and a mutant protein results that is involved in the transformation of normal cells to cancer cells. The actual function by which the protein acts in the signal pathway is not known. However it is known that they act as a switch, undergoing a cycle involving the exchange of guaninosine nucleotides in the binding site. This thesis uses computer simulations to study the energetics of this binding, with the long term aim of developing a drug to inhibit the transforming activity of the oncogenic protein. To begin with, a model of the protein based on a crystal structure is built. Using Molecular dynamics the motion of this model is studied. A possible mechanism by which one half of the nucleotide cycle could be induced is investigated, with the result that phosphorylation of the protein may be involved. The main part of the thesis is then devoted to using the free energy perturbation (FEP) method to calculate the difference in Gibbs binding free energy between the nucleotides in the protein. Using histamine as a model, a method of dealing with charged, flexible molecules is developed; namely the inclusion of a reaction field and comprehensive conformational analysis. The results from the associated calculations are seen to be very close to experimental data. The same procedures are then applied to the much more complex ras: nucleotide system with less successful results, the reason for which is mostly due to the restriction of limited computer resources to tackle such a problem. The conclusion is that given the resources and by using the techniques developed in this thesis, this type of calculation is a feasible way to study such systems.
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Jakob, Valentin [Verfasser]. "Inhibition of protein-nucleic acid interactions mediated by viral and bacterial proteins via fragments and peptides / Valentin Jakob". Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2021. http://d-nb.info/1240674155/34.
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Chakrabarti, Partha Pratim. "Biochemical and biophysical analysis of the GTPase activating proteins of the small guanine nucleotide binding protein Rap1 and RheB". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975809598.
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Stains, Cliff. "Methods for the Detection of Protein-Nucleic Acid and Protein-Protein Interactions". Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194834.
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We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 10^6-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.We envision that the assay platforms described herein will find applications in the rapid detection of nucleic acid sequences, protein identities, and relative protein abundances in the laboratory and clinic.
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Chen, Baoshan. "Encapsidation of nucleic acids by cucumovirus coat proteins /". Title page, contents and summary only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phc5183.pdf.
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Toro, Imre. "Crystal structures of two nucleic acid-binding proteins". Thesis, Open University, 2000. http://oro.open.ac.uk/58089/.
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The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.
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Smyth, Clare Patricia. "Oligomerisation of the chromatin-structuring protein, H-NS". Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325351.
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Underwood, E. A. "Nucleotide regulation of AMP-activated protein kinase". Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1382013/.
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AMP-activated protein kinase (AMPK) acts as the cell’s master energy regulator, sensing and maintaining the concentration of ATP in a narrow range irrespective of energy demand. This kinase has received significant attention as a drug-target for type-2 diabetes, obesity and cancer. Although historically the AMP:ATP ratio has been considered the signal for AMPK activation, we have recently demonstrated that ADP is likely to be an important physiological regulator of AMPK in both mammals and yeast. The binding of adenine nucleotides and staurosporine to the full-length alpha1beta1gamma1 heterotrimer, both phosphorylated and unphosphorylated, is described. Binding was monitored through displacement of fluorescently labelled nucleotides (coumarin-AXP), either via direct coumarin excitation or Forster Resonance Energy Transfer (FRET) in which tryptophan residues were excited. Mg.ATP was found to bind more weakly than ADP, a feature which is likely key to AMPK regulation. A Nicotinamide adenine dinucleotide (NADH) coupled spectrophotometric assay was used to monitor AMPK kinetics and its regulation by nucleotides. NADH binds at Site-1, within the gamma-subunit, and competes with allosteric activation by AMP, but not the protective effect of AMP/ADP against alpha-T172 dephosphorylation. Therefore it seems that AMP binding at Site-1 mediates allostery whilst AMP/ADP binding at Site-3 affords protection against dephosphorylation. In order to explore this idea further, AXP binding constants were used to model binding site occupancies over the concentration ranges used in vitro. The modelling demonstrates that, in vitro, Site-1 is occupied by AMP and Site-3 by AMP/ADP in a manner consistent with their assigned regulatory functions. This modelling study was also extended to consider in vivo binding site occupancy. It was important to verify that coumarin-ADP bound in a homologous fashion to ADP, specifically in the same exchangeable binding pockets. X-ray crystallography was used to determine the structure of a truncated form of AMPK in complex with coumarin-ADP. This structure is compared to an ADP-bound form. SNF1 is the Saccharomyces cerevisiae AMPK ortholog. The binding of nucleotides to SNF1, and its regulation by ADP, was also characterised. As with the mammalian enzyme AXP bound at two exchangeable sites, and interacted with Mg.ATP more weakly than ADP.
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Horowitz, Eric D. "Intercalator-mediated assembly of nucleic acids". Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/33937.
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The RNA World hypothesis suggests that RNA, or a proto-RNA, existed in an early form of life that had not yet developed the ability to synthesize protein enzymes. This hypothesis, by some interpretations, implies that nucleic acid polymers were the first polymers of life, and must have therefore spontaneously formed from simple molecular building blocks in the "prebiotic soup." Although prebiotic chemists have searched for decades for a process by which RNA can be made from plausible prebiotic reactions, numerous problems persist that stand in the way of a chemically-sound model for the spontaneous generation of an RNA World (e.g., strand-cyclization, heterogeneous backbones, non-selective ligation of activated nucleotides). The Molecular Midwife hypothesis, proposed by Hud and Anet in 2000, provides a possible solution to several problems associated with the assembly of the first nucleic acids. In this hypothesis, nucleic acid base pairs are assembled by small, planar molecules that resemble molecules which are known today to intercalate the base pairs of nucleic acid duplexes. Thus, the validity and merits of the Molecular Midwife hypothesis can be, to some extent, explored by studying the effects of intercalation on the non-covalent assembly of nucleic acids.
In this thesis, I explore the role of the sugar-phosphate backbone in dictating the structure and thermodynamics of nucleic acid intercalation by using 2′,5′-linked RNA intercalation as a model system of non-natural nucleic acid intercalation. The solution structure of an intercalator-bound 2′,5′ RNA duplex reveals structural and thermodynamic aspects of intercalation that provide insight into the origin of the nearest-neighbor exclusion principle, a principle that is uniformly obeyed upon the intercalation of natural (i.e. 3′,5′-linked) RNA and DNA. I also demonstrate the ability of intercalator-mediated assembly to circumvent the strand-cyclization problem, a problem that otherwise greatly limits the polymerization of short oligonucleotides into long polymers. Together, the data presented in this thesis illustrate the important role that the nucleic acid backbone plays in governing the thermodynamics of intercalation, and provide support for the proposed role of intercalator-mediated assembly in the prebiotic formation of nucleic acids.
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Butler, Thomas. "Nanopore analysis of nucleic acids /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9674.
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Vickers, Mark F. "Characterization of bacterial and mammalian recombinant nucleoside transport proteins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0009/NQ59688.pdf.
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Holder, Mark Travis. "Using a complex model of sequence evolution to evaluate and improve phylogenetic methods". Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3037500.
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Zhao, Dengke. "Quantitative Modeling of Protein - Nucleic Acid Interactions". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534755895588153.
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Annoni, C. "NEW DIMENSIONS INTO PROTEIN-NUCLEIC ACIDS INTERACTIONS". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232405.
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In the late 19th century, scientists microscopically observed the association of proteins with DNA strands. Since then, researchers have used a variety of in vitro and in vivo assays to demonstrate that proteins interact with DNA and RNA to influence the structure and function of the corresponding nucleic acid. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination and RNA processing and translocation continues to revolutionize our understanding of cell biology, normal cell development and the mechanisms of disease. Furthermore, there is the potential for constructing new molecules with excellent functionalities by assembling the very simple elements and components that are the functional subunits of these natural biopolymers. In this work of thesis we attempted making a new step forward a better knowledge of protein-nucleic acid biopolymers, focusing on two complexes of strong interest. One of the important factors for determining the expression of the function is the structure of the biopolymer. Clarification of the relationship between the structure and the function of biopolymer is of crucial importance to the development of methods for constructing tailor-made functional molecules. The first goal of my thesis focalized on the obtainment of a structural model of a well-known protein-DNA complex: Maf DNA binding domain and it DNA target (T-MARE was used in this study) with the aim of outlining a strategy for the obtainment of activity modulators. Both in silico simulations and experimental studies were carried out leading to the definition of a modus-operandi based on a disorder-order transition of the complex which is commendatory for the protein activation. On the other hand, a number of artificial enzymes have been recently constructed by using the molecular design based on structural information, screening methods that utilize libraries or by a combination of these two methods. However, the above mentioned approaches use single proteins or single nucleic acid as the structural unit, and the activity of the constructed artificial enzymes is remarkably lower in many cases, than that of the native enzymes. The construction of functional complexes (rather than a single biopolymer) as scaffold can be considered as one potential solution to these drawbacks. Further in my studies, using the HIV-Rev peptide and RRE (Rev Responsive Element) RNA complex as a scaffold, for which the tridimensional structure was fully characterized, the assemble of ribonucleopeptidic fluorescent sensors was accomplished in a stepwise manner. In this method, a randomized nucleotide sequence was introduced into the RNA subunit of RNP to construct a RNP library on which the in vitro selection method was applied. In the second step, the Rev peptide was modified with the fluorophore without altering the affinity and specificity of the RNP receptor. In the absence of a ligand for RNP, fluorescence emission was effectively quenched in the RNP complex, but recovered upon ligand binding. RNP sensors were thus created, as the ligand-binding event can be monitored by measurement of the fluorescence signals.
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Adair, Jennifer Eileen. "Molecular and genetic influence of HMGA1 proteins on nucleotide excision repair". Online access for everyone, 2005. http://www.dissertations.wsu.edu/Dissertations/Fall2005/j%5Fadair%5F120605.pdf.
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Guo, Maolin. "Proteins and nucleic acids as targets for titanium(IV)". Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/13967.
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TiIV compounds have pronounced anticancer, antiviral and antibacterial activities, and titanocene dichloride (TDC) is currently on phase II clinical trials as an anticancer drug. However, the biological chemistry and mechanisms of action of TiIV are poorly understood. Proteins and nucleic acids are expected to be biological targets for TiIV. Human transferrin (hTF) is a bilobal serum glycoprotein (80 kDa) which transports FeIII to cells via receptor-mediated endocytosis. A structurally similar periplasmic iron binding protein (FBP, 34 kDa) is present in some pathogenic bacteria and is required for virulence. In this thesis, the aqueous coordination chemistry of TiIV with the phenolate ligand N,N'-ethylene-bis-(o-hydroxyphenylglycine)(H4ehpg) was investigated as a model for Ti-hTF (or FBP) interactions. TiIV forms 7-coordinae monomer (rac) and dimer (meso) complexes with H4ehpg (rac + meso) with novel stereo-selectivity. 1H and 31P NMR studies show that TDC binds selectivity to H4ehpg at neutral pH, but preferentially to adenosine triphosphate (APT) at pH values below 5; TiIV transfers from TiIV-ehpg to ATP at acidic pH values. The interactions of TDC with hTF and that of Ti2-hTF with ATP have characteristics which could allow transferrin to act as a mediator for titanium delivery to tumour cells. TDC reacts rapidly with apo-hTF under extra-cellular conditions and binds in the specific FeIII sites with release of the Cp and Cl ligands. TiIV is readily released from Ti2-hTF at endosomal pH (ca 5.0) and in the presence of ATP. Ti2hTF competes effectively for cell uptake of 59Fe-hTF into BeWo cancer cells. TDC binds strongly to the phosphate group of nucleotides in aqueous solution and TiIV binds to the phosphodiester groups of nucleotides in the less polar solvent N,N-dimethylformamide. This behaviour contrasts with that of the anticancer drug cisplatin which binds mainly to N-sites of nucleobases, and may account for the intracellular localisation behaviour of TiIV drugs.
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Moran-Jones, Kim. "hnRNPs A2 and A3 : nucleic acid interactions /". St. Lucia, Qld, 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17983.pdf.
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Moodie, Stuart Leslie. "Molecular recognition : computational studies on protein nucleotide interactions". Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362460.
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Raper, Jayne. "Guanine nucleotide binding protein function in T.B. Brucei". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305533.
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Fitzgerald, Amanda Ann. "Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation". eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/341.
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Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology.
The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed.
These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.
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Law, Wing-lun, i 羅永倫. "Expression, purification and preliminary x-ray crystallographic studies of two nucleotide binding proteins". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46939118.
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Pickford, Christopher. "Mechanism of nucleotide exchange by guanine nucleotide exchange factors on the Ras superfamily of small G-proteins". Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325813.
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Lee, Semin. "Molecular characterization of protein-nucleic acid interfaces : applications in bioinformatics". Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609284.
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