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1
Guo, Xiangxue. "Biochemical and Bioinformatics Analysis of CVAB C-Terminal Domain." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/biology_diss/3.
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Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.
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Carvalho, Maria João Marques de. "Characterization of a C-terminal domain from eag potassium channel." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/4343.
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Mestrado em Métodos BiomolecularesDomínios que ligam nucleotideos cíclicos (CNBD) regulam muitas vias de sinalização em células procarióticas e eucarióticas. Os ligandos AMP cíclico ou GMP cíclico ligam-se a estes domínios e induzem uma alteração conformacional que é propagada ao domínio efector, como uma cinase ou um canal iónico. Os canais de potássio da família ether-a-go-go (EAG) estão envolvidos em muitos processos fisiológicos que incluem repolarização cardíaca e neuronal, proliferação tumoral e secreção de hormonas. Estes canais são tetraméricos e cada subunidade inclui seis hélices transmembranares e dominios citoplasmáticos em N- e C-terminal. O domínio em C-terminal tem homologia com domínios que ligam nucleotídeos cíclicos mas foi demonstrado que os canais EAG não são afectados por nucleotídeos e o domínio não liga nucleotideos. O objectivo deste projecto foi resolver a estrutura de um domínio C-terminal de um canal EAG por cristalografia de raios-X e compreender o seu papel funcional. Determinei a estrutura de um destes domínios à resolução de 2,2 Å; a estrutura tem a topologia de um CNBD mas a cavidade de ligação apresenta várias diferenças relativamente à de domínios que ligam nucleotideos cíclicos. Mais ainda, os canais EAG são inibidos por calmodulina e há dois locais de ligação de calmodulina a seguir ao CNBD. A estrutura mostrou que um destes locais se encontra sobreposto com uma região do domínio levantando a possibilidade da calmodulina regular o canal através da alteração conformacional do domínio C-terminal dos canais EAG. Esta possibilidade começou a ser explorada com recurso a ensaios de cross-linking químico e espectroscopia de fluorescência.
Cyclic nucleotide binding domains (CNBD) are regulatory domains that participate in many signaling pathways in prokaryotic and eukaryotic cells. The ligand cAMP or cGMP binds these domains and induces a conformational change that is propagated to an effector domain, like a kinase or an ion channel. The ether-a-go-go (EAG) potassium channel family is involved in important physiological roles that include cardiac and neuronal repolarization, tumor proliferation and hormone secretion. These channels are tetramers, where each subunit includes six transmembrane helices and N- and C-terminal cytoplasmic domains. The C-terminal domain has strong homology to CNBDs but it has been demonstrated that EAG channels are not affected by cyclic nucleotides and that the domain does not bind nucleotides. The ultimate goal of this project was to solve the structure of an EAG family C-terminal domain by X-ray crystallography and to understand its functional role. I have determined the structure of one of these domains at 2.2 Å; the structure has the canonical CNBD fold but it shows a ligand pocket that has several differences relative to a cyclic nucleotide binding site. Furthermore, EAG currents are inhibited by calmodulin binding and there are two calmodulin binding sites C-terminal to the CNBD. The structure reveals that one of these sites overlaps with a region of the domain raising the possibility that calmodulin affects channel function by changing the EAG C-terminal domain conformation. I have conducted preliminary tests on this hypothesis by using biochemical cross-linking experiments and fluorescence spectroscopy.
FCT
FCOMP-010124-FEDER-007427/PTDC/QUI/66171/2006
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Miller, Wayne. "Structural characterisation of the prokaryotic sodium channel C-terminal domain." Thesis, Birkbeck (University of London), 2015. http://bbktheses.da.ulcc.ac.uk/140/.
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Since the discovery of the first prokaryotic voltage gated sodium channel (Nav) in 2001, prokaryotic Navs have been a high priority target for structural study. Prokaryotic Navs are of interest as a model system due to their homology to eukaryotic Navs, which are high value drug development targets for their roles in pain perception and neural function. While prokaryotic Navs have function and pharmacology distinct from their eukaryotic homologues, understanding their structure holds implications for drug development and for understanding diseases stemming from neuronal dysfunction. However, Navs have historically been challenging targets for structural study, resisting attempts at crystallisation until recently. In this study, expression, purification, and characterisation of a chimera of the NavBh channel and the ligand gating RCK domain from the prokaryotic potassium channel MthK has been performed. It was hypothesised that the addition of the RCK domain would improve the channel’s crystallisation potential, and create a ligand gated Nav for functional characterisation. Electrophysiological studies demonstrated that the RCK domain was capable of gating NavBh, however the chimera had reduced solubility, indicating that this chimeric fusion was not an ideal target for structural study due to low purification yields. Following this, and in light of recent studies that suggested the structure of the prokaryotic Nav C-terminus had a role in channel function, structural analysis of the C-terminus of a prokaryotic Nav homologue cloned from Bacillus alcalophilus has been performed. Synchrotron radiation circular dichroism analysis of serial C-terminal truncations demonstrated the structure of the NsvBa C-terminus consists of a helical region connected to the channel pore by a disordered neck region, despite conflicting bioinformatics predictions. This offers further support for the hypothesis that in functional Navs, the C-terminus consists of a disordered neck region connecting a coiled-coil to the base of the pore, which acts as a spring to assist in channel gating and inactivation.
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Benetti, Federico. "Structural studies on the C-terminal domain of human PMCA1b." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425143.
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Plasma Membrane Calcium ATPases (PMCAs) are P-type pumps involved in calcium homeostasis. Their 3D structures are still unknown but a possible topology has been predicted. PMCAs are predicted to have a cytosolic N-terminal domain, a cytosolic C-terminal domain (regulatory domain), ten transmembrane segments and two cytosolic loops called transduction domain and catalytic domain that connect the 2nd and the 3rd, the 4th and the 5th transmembrane segments respectively. Several mechanisms are responsible of their activation: interaction with Ca2+-calmodulin, interaction with acidic phospholipids and fatty acids, phosphorylation with kinases A and C, proteolysis by calpain and oligomerization. All activation mechanisms decrease the Km values, in particular the oligomerization brings this value around at the value of cytosolic calcium concentration present in the resting cells (50-100 nM).
The C-terminal domain is a structural motif that distinguishes PMCAs from all other P-type pumps. It is also the target of all activators and activation mechanisms. In this study we have described the secondary structure and tertiary structure at low
resolution of the C-terminal regulatory domain of the human PMCA isoform 1b. We have found that the domain forms aggregates by intermolecular interactions. Moreover, we have studied the reversibility of the oligomerization process and
found the best conditions to stabilize the C-terminal domain in the monomeric form. These conditions imply the presence of the lipid mimetic SDS at critical micellar concentration. A structural reconstruction based on Small Angle Neutron Scattering experiments provides a low resolution structure where the C-terminal domain has an hourglass shape. The central cross section compatible with that of an ?-helix. This
part could correspond at the ?-helix of the C28W calmodulin binding region while the downstream and upstream regions could be random coil as also predicted by PSIpred. Binding experiments between the C-terminal domain and the Ca2+-
calmodulin have been carried out. The aim was to study whether in a phospholipid mimetic system necessary to stabilize the monomeric form, such as sodium dodecyl sulphate, this domain can still interact with calmodulin. The phospholipid mimetic system that stabilize the domain in the monomeric form prevent its binding with Ca2+-calmodulin. The results suggest that a different aggregation state of the PMCAs
exist in diverse membrane rafts: membrane rafts rich in uncharged or zwitterionic phospholipids could contain PMCAs in oligomeric form while membrane rafts rich in acidic phospholipids could contain PMCAs in monomeric form.
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Adu-Bobie, Jeanette. "Characterisation of the C-terminal domain intimin from enteropathogenic Escherichia coli." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300436.
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Ragan, Timothy James. "Regulation of S6K1 Protein Kinae Activation by its C-Terminal Autoinhibitory Domain." Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/125.
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Signal transduction kinases lie at the heart of the cell's ability to respond to environmental cues. These kinases are typically controlled by post-translational modification, most commonly by phosphorylation. S6K1alphaII is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (T389) regions of its catalytic kinase domain. In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID, residues 399-502), which inhibits T-loop and hydrophobic motif phosphorylation. Autoinhibition is relieved upon multi-site Ser-Thr phosphorylation of the AID by MAP kinase(s). We developed an optimized PCR-based gene synthesis method, which I utilized to build expression constructs for the AID alone as well as the kinase domain and full length S6K1alphaII. A fully activated form of S6K1alphaII was purified from Sf9 cells by co-expression with PDK1, and was used for in vitro analysis of the signaling pathway. AID was successfully purified in a soluble form from E. coli despite the fact that PONDR analysis predicted a highly disordered structure. Aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC spectra and far UV CD data showing a lack of secondary structure, confirmed that purified recombinant AID is largely unfolded. Despite this, addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1 and inhibition was decreased when the tetraphospho-mimic mutant AID(D2ED) was used. These studies, along with the reagents produced by them, will allow for further exploration of the emerging field of disordered regulatory domains.
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Panagiotidou, P. "Cloning, expression and structural studies on the C-terminal domain of procollagen C-proteinase enhancer (ctPCPE)." Thesis, University of Kent, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405998.
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Al-Ali, Hassan. "Regulation of PDK1 Protein Kinase Activation by Its C-Terminal Pleckstrin Homology Domain." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/381.
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Phosphoinositide-dependent protein kinase-1 (PDK1) plays an integral role in signaling cellular growth and proliferation, one that's dependent on its ability to autophosphorylate Ser-241 in its T-loop. This process appears to have a strict requirement for its C-terminal pleckstrin homology (PH) domain. Thus, the overall objective of this work was to determine the mechanism by which the PH domain induces an active kinase conformation in unphosphorylated PDK1, capable of Ser-241 autophosphorylation. First, computational modeling and protein cross linking studies were combined with site-directed mutagenesis and kinetic assays in order to provide initial assessment of how the PH domain scaffolds Ser-241 autophosphorylation. A significant number of contacts were identified between the enigmatic "N-bud" region of the PH domain and the kinase domain. Specifically, these studies implicated Glu-432 and Glu-453 of the N-bud region of the PH domain that bind and serve as mimics of the phosphorylated Ser-241 in the T-loop and the phosphorylated C-terminal tail of PDK1 substrates, respectively. Next, a novel method for protein trans-splicing of the regulatory and catalytic kinase domains of PDK1 was developed. The method utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. The cross-reacting KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH fusion constructs generated full length spliced-PDK1 with kobs = (2.8 +- 0.3) x 10-5 s-1. Finally, NMR was used to further characterize the structural and dynamical properties of the PH domain in both its isolated form and in full length PDK1. Whereas, it was not possible to obtain chemical shift assignments of any backbone or side chain nuclear resonances, methods were optimized for 2H,13C,15N-isotopic labeling of the recombinant PH domain. Furthermore, the protein trans-splicing method was significantly improved and utilized for segmental isotopic labeling of the PH domain in full length PDK1. These new findings and developments may provide specific insight and technological improvements towards future studies aimed to better understand and target autoinhibited conformations of PDK1 for translational purposes.
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Chapman, Rob. "A Functional Analysis of the RNA Polymerase II Large Subunit C-Terminal Domain." Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-11049.
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Ray, Pampa. "DNA binding studies of the transcriptional activator NifA and its c-terminal domain." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393779.
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Chattopadhyay, Anasuya. "Study of the C-terminal domain of variant surface glycoproteins from African trypanosomes." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619987.
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Cook, Carma Oshea Goodwin Douglas C. "Role of distant, intrasubunit residues in catalase-peroxidase catalysis tracing the role of gene duplication and fusion in enzyme structure and function /." Auburn, Ala, 2009. http://hdl.handle.net/10415/1733.
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Boeing, Stefan. "Functions of Mediator and the RNA Polymerase II C-terminal Domain in Transcription Initiation." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-97050.
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Yang, Kimberly M. "Binding Studies of Membrane Receptor CD47 with the C-terminal Domain of Thrombospondin-1." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/146078.
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Secreted glycoprotein Thrombospondin-1 (TSP1), among its many functions, binds to membrane receptor CD47 in order to downregulate the nitric oxide pathway via inhibition of soluble guanylate cyclase (sGC). Physiologically, this process is anti-angiogenic and also affects vasodilation, platelet aggregation and tissue ischemia. Aside from the key players TSP1, CD47 and sGC, little is known about the mechanism of this pathway. This project seeks to understand the binding interaction between trimeric TSP1 and transmembrane receptor CD47. It is hypothesized that the C-terminal binding domain of TSP1 (E3CaG1) and the N-terminal extracellular domain of CD47 (CD47-NT) are sufficient to mediate binding. E3CaG1 was expressed and purified from Sf9 insect cells while CD47-NT was expressed and refolded from inclusion bodies in E. coli. Binding was tested via co-immunoprecipitation experiments utilizing both nickel and GST affinity resins. It was found that the GST- and His-tagged constructs of CD47-NT did not show any interaction with E3CaG1. While E3CaG1 has been shown to be fully active, further work is underway to determine whether CD47-NT is also properly folded and active.
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Bao, Xiaomin. "Functional analysis of the C-terminal domain in the Drosophila JIL-1 histone H3 kinase." [Ames, Iowa : Iowa State University], 2008.
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Soegaard, Teit Max Moscote. "The RNA polymerase II C-terminal domain : its phosphorylation and interaction with the mediator complex." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445856/.
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The submitted thesis describes a biochemical investigation within the field of gene transcription. In yeast (Saccharomyces cerevisiae ) and the metazoans, the conserved RNA polymerase II (RNAPTJ) protein complex carries out regulated transcription. The C-terminal domain (CTD) of Rpbl (the largest subunit of RNAPII) consists of 26 hepta-peptide (YSPTSPS) repeats that are targeted by specific CTD-kinases and - phosphatases to change the phosphorylation of Ser2 and Ser5 resulting in two electrophoretically different forms of RNAPII - a fast migrating hypo-phosphorylated form and a slower migrating hyper-phosphorylated form. The changing patterns of phosphorylation modulate the interaction of multiple factors involved in transcription initiation, RNA processing, transcription elongation and RNAPII ubiquitylation throughout the transcription cycle. Prior to transcription a preinitiation complex (PIC) is assembled on the promoters of regulated genes. A plethora of regulatory pathways converge on a protein complex named Mediator - a central, functionally conserved protein complex that incorporates into the PIC to physically interact with RNAPII and which can only interact with hypo-phosphorylated RNAPII. Mediator plays the crucial role of relaying repressive and activating signals from transcription factors bound on the promoter to RNAPII poised at the beginning of the coding sequence of the gene. As Mediator is only found associated with promoter regions on chromatin RNAPII must dissociate from Mediator when transcription initiates and promoter escape occurs. The mechanism leading to dissociation of RNAPII from Mediator is unknown. A large conformational change in Mediator accompanies the association with RNAPII and the interaction is dependent both on the C-terminal domain of Rpbl (CTD) and of other members of the RNAPII complex. If hypo-phosphorylation is needed for Mediator to interact with RNAPII does hyper-phosphorylation lead to the dissociation of the two complexes Are other factors needed to bring about the dissociation of the holopolymerase complex By using purified forms of Mediator and holopolymerase and purified specific CTD kinases the work presented addresses these questions in vitro.
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LAL, KANHAYA. "STRUCTURE-BASED DESIGN OF GLYCOMIMETIC LIGANDS FOR THE N-TERMINAL DOMAIN OF BC2L-C LECTIN." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/893211.
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The prevalence of drug-resistant infections has challenged the existing treatment regimen using antibiotics. There is a need to discover and employ alternative and complementary therapies to counteract these life threatening infections. In fast few decades, the use of anti-adhesion molecules targeting virulence factors such as lectins has been proven an attractive approach to counteract the infections by disarming the pathogens. This thesis work aimed to design glycomimetic antagonists of the N-terminal domain of the BC2L-C lectin (BC2L-C-nt) from the drug-resistant pathogen known as B. cenocepacia. We employed a fragment-based approach to design glycomimetic antagonists of the target protein (BC2L-C-nt). The initial studies were focused towards the binding site prediction and target evaluation by computational tools which identified additional druggable regions near the fucoside binding site in BC2L-C-nt. These additional regions have been explored further to evaluate the druggability by employing virtual screening of a small fragment library. This identified an interesting region (region ‘X’) that could host the drug-like fragments by establishing some key interactions. These interactions with the lectin have been confirmed using a group of biophysical techniques, including X-ray crystallography. Remarkably, the binding mode of one of the fragment (KL3) has been validated by X-ray crystallography at high resolution confirming the ability of site X to host drug-like fragments. Further, the fragments have been chemically connected to the fucose core to obtain high-affinity bifunctional glycomimetic ligands. Interestingly, the crystal complexes of BC2L-C-nt with two bifunctional glycomimetic ligands again confirmed the druggability of the identified site, thus also validated the computational predictions. Hence, the first generation of glycomimetic ligands with binding affinities in micromolar range have been successfully designed. These glycomimetics provide further opportunities to design high-affinity ligands. Future studies based on structure-based approaches and robust synthetic routes to synthesize glycomimetics can lead towards the high-affinity ligands as anti-adhesive agents against B. cenocepacia.
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Lui, Winnie Wing-yin. "AP-3 subunit interactions and proteins that interact with the C-terminal domain of γ-adaptin." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621195.
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Gnesa, Eric Henry. "The conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/771.
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Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp 1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the post-spin draw ratios of the fibers . Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility, but maintained constant toughness.
Wide-angle X-ray diffraction studies indicate that post-drawn fibers containing the Cterminal domain of TuSp 1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to non-tagged recombinant dragline silk proteins spun from equivalently sized proteins.
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Morris, Benjamin L. "Understanding and targeting the C-terminal Binding Protein (CtBP) substrate-binding domain for cancer therapeutic development." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4434.
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Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.
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Baker, Ruletha Deon Goodwin Douglas C. "Roles of an 'inactive' domain in catalase-peroxidase catalysis modulation of active site architecture and function by gene duplication /." Auburn, Ala., 2006. http://repo.lib.auburn.edu/2006%20Fall/Dissertations/HARTFIELD-BAKER_RULETNA_19.pdf.
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Colombo, Maria Carola. "On the structural stability of the free and metal-loaded c-terminal domain of the prion protein /." [S.l.] : [s.n.], 2006. http://library.epfl.ch/theses/?nr=3592.
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Roether, Susanne. "Functional Analysis of the RNA Polymerase II C-terminal Domain Kinase Ctk1 in the Yeast Saccharomyces cerevisiae." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-73014.
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Mitra, Sharmistha. "Ubiquitin Modulates Tollip's PtdIns(3)P Binding and Dissociates the Dimeric State of C-Terminal Cue Domain." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/51151.
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Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. In this study, we describe for the first time how ubiquitin controls the function of the Toll-interacting protein (Tollip), which is an effector protein in the innate immune signaling pathway and an adaptor protein for endosomal trafficking. We have demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides with moderate affinity. Remarkably, we found that ubiquitin modulates Tollip's lipid binding. We have observed an ubiquitin dose-dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to discover that the Tollip C2 domain is a novel ubiquitin-binding domain. In addition, we have biophysically characterized the association of the Tollip CUE domain to ubiquitin and compared it with Tollip C2 domain-ubiquitin binding. The Tollip CUE domain reversibly binds ubiquitin with affinity higher than C2 domain and at a site that overlaps with that corresponding to the Tollip C2 domain. We have also found that ubiquitin binding to dimeric Tollip CUE domain induces a drastic conformational change in the protein, leading to the formation of a heterodimeric Tollip CUE-ubiquitin complex. These data suggest that ubiquitin binding to the Tollip C2 and CUE domains and ubiquitin-mediated dissociation of CUE dimer reduces the affinity of the Tollip protein for endosomal phosphoinositides, allowing Tollip's cytoplasmic sequestration. Overall, our findings will provide the structural and molecular basis to understand how Tollip works inside the cell and commit itself to cytosolic signalling or endosomal trafficking in a ligand dependent manner.Ph. D.
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Brunecky, Roman. "Structural studies of the C-terminal domain of the dopamine transporter and its interaction with [alpha]-synuclein /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 105-113). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Wang, Songtao. "The structural requirements and mechanisms for high affinity CA2+ binding and MG2+ binding to the C-terminal domain of cardiac troponin C /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487862972134785.
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Oladosu, Oyindamola. "Structures et fonctions du domaine C-Terminal de l'intégrase du VIH-1." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ025/document.
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L’Integrase du VIH est une ADN recombinase catalysant deux réactions qui permettent l'intégration de l'ADN viral dans l'ADN hôte. L’intégrase du VIH comprend 3 domaines : N-terminal impliqué dans la réaction de « 3' processing » et le transfert de brin, le domaine catalytique contenant le site actif et le domaine C-terminal liant l'ADN non-spécifiquement (CTD). Des recherches récentes mettent en évidence l'importance du CTD dans la liaison avec d'autres protéines virales comme la transcriptase inverse. Le but de la thèse était de comprendre les rôles et l'importance du domaine C-terminal de l’intégrase dans deux contextes : l'intégration dans la chromatine et la coévolution, avec l'objectif de comprendre le rôle de la multimerisation dans la fonction de l’intégrase. Globalement, les résultats de mon projet indiquent que l'IN-CTD joue un rôle important, en contribuant à la formation de multimères d'ordre supérieur importants pour la fonction de l’INHIV Integrase is a DNA recombinase that catalyzes two endonucleolytic reactions that allow the viral DNA integration into host DNA for replication and subsequent viral protein production. HIV Integrase consists of 3 structural and functional domains: The N-terminal zinc domain involved in 3’ processing and strand transfer, the catalytic core domain which contains the active site, and the C-terminal domain that binds DNA non- specifically. Recent research highlights the importance of the CTD in binding with other viral proteins such as Reverse Transcriptase. The aim of the thesis was to understand the roles and importance of the C-terminal domain of HIV-1 Integrase in two contexts: chromatin integration, and co-evolution, with the overall purpose of understanding the role of multimerization in IN function. Overall, results from my project indicate that the IN-CTD plays an important role, by contributing to the formation of higher order multimers that are important for IN functionality
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Fan, J. "Production and characterisation of specific antibodies to regions of the C-terminal domain of cytoplasmic dynein heavy chain." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598935.
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Cytoplasmic dynein (CD) is a multisubunit microtubule (MT) motor protein that transports vesicles and other particles along cellular microtubules (MTs). The aim of the work in this thesis is to better understand the relationship between dynein's molecular structure and function, especially the mechanics of CD-MT interactions. Two approaches have been used. (1) Using recombinant techniques, a number of overlapping domains (each around 30 KDa) of DHC, downstream of the coiled coil stalk, were expressed in bacteria and their in vitro MT-binding properties were characterised by cosedimentation with MTs. (2) Using immuno-techniques, a number of specific antibodies to the expressed proteins or to synthetic peptides have been produced and characterised. Two good anti-peptide antibodies were further used to assay their effects on native dynein-driven MT motility in vitro. The results showed that one expressed protein, termed CD-H2, bound to MT's in vitro and that an antibody against 16 residues outside this region did not. The conclusion is that the region of the sequence involved in forming the surface that interacts with MTs probably extends at least 500 residues downstream of the known MT-binding sites on the coiled-coil stalk and plays an essential role in MT-DHC interaction during motility. Some preliminary studies by immuno-electron microscopy indicate that it will be possible to locate this and other functional regions on the observed structure of native CD molecules.
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Fry, A. C. "A novel mutation in SLC4A1 causing distal renal tubular acidosis : an investigation of the AE1 C-terminal domain." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599245.
Full textAbstract:
This research identifies a novel AE1 mutation in a newly-referred family where dRTA segregates with disease through a four-generation pedigree, as determined by DNA sequencing and specific restriction enzyme digestion. This mutation – AE1-M909T – lies within the last four residues of the tail, in a potential PDZ-binding domain. AE1-M909T expression in Xenopus oocytes confirmed preserved anion exchange function. To investigate the mechanism of disease, N-terminal GFP-tagged AE1 was stably expressed in a variety of cell types in HEK293 and non-polarised MDCK cells, wild-type (AE1wt) and AE1-M909T both reach the cell surface. In polarised MDCK cells, GFP-tagged AE1wt has normal basolateral residency. In marked contrast, the mutant protein is aberrantly targeted, appearing at the apical membrane in addition to preserved basolateral presence. Antibody-labelling assays demonstrated post-synthetic AE1wt traffic direct to the basolateral membrane without any apical passage. In contrast, AE1-M909T traffics directly to both cell surfaces, thus implying gain of an apical targeting signal (plus persistent basolateral signals): this non-polarised expression will cause failure of α-IC function and dRTA. This novel SLC41 mutation causes AE1 mistargeting through acquisition of an apical targeting signal. The results suggest that basolateral targeting motifs are concentrated upstream of the extreme C-terminus of AE1, but that this micro-domain is important for normal biosynthetic traffic to the cell surface, potentially through PDZ-based interactions.
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Martel, Marc-Andre´. "Role of the NR2 subunit composition and intracellular C-terminal domain in N-methy-D-aspartate receptor signalling." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4228.
Full textAbstract:
N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ionotropic receptors. When activated, NMDARs let extracellular sodium and calcium ions enter neurons. This calcium influx, depending on its duration, intensity and the presence of nearby signalling proteins can signal to synaptic plasticity. Additionally, physiological NMDAR activity promotes pro-survival cascades and gene transcription, whereas both lack of activation and overactivation of these receptors trigger pro-death signals. Several neurodegenerative pathologies such as stroke/ischemia and Alzheimer’s disease are thought to involve NMDAR overactivation, so-called “excitotoxicity”, but since NMDARs are important for normal neuronal physiology, potential therapeutical approaches needs to go beyond simple antagonism. Here, we studied the receptor subunit composition and the molecular cascades downstream of the receptor activation to try and isolate the pro-death pathways in NMDAR-mediated excitotoxicity. We found that the NR2 subunit composition did not dictate the type of NMDAR-mediated signals, as receptors comprised of NR2B subunits were able to signal to death, survival and plasticity. However, we also found that the intracellular tail of the NR2B subunit was more efficient at triggering neuronal death compared to the NR2A C-terminus, which suggests that different pro-death signalling complexes are associated to each subunit. Two pro-death signals, the p38 and c-Jun N-terminal kinase (JNK) cascades, are key mediators of neuronal excitotoxicity. In a non-neuronal cell line, NMDAR-mediated cell death could be reconstituted but was found to rely solely on JNK and not p38. This was due to the lack of pro-death signals from the NR2B-PDZ domain, a cytoplasmic interacting domain which forms a signalling cassette with the neuronal proteins PSD-95 and neuronal nitric oxide synthase. This PDZ-ligand recruits the p38 cascade in neurons, but was absent in non-neuronal cells. The pro-death p38 pathway could be inhibited in neurons by disrupting the PDZ domain interactions, which protects against excitotoxicity. This disruption was not affecting normal synaptic transmission, potentiation or survival signalling, suggesting that this could be a therapeutically viable avenue. Thus, this work has expanded the understanding of how NMDAR subunits and their cytoplasmic domains mediate signalling leading to a variety of cellular outcomes; a crucial point for the development of a strategy specifically targeting NMDAR- mediated pro-death signalling.
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Poonsiri, T. "Structural study of the C-terminal domain of non-structural protein 1 and capsid protein from Japanese encephalitis virus." Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3021941/.
Full textAbstract:
Japanese encephalitis virus (JEV) is a mosquito-transmitted Flavivirus that is closely related to other emerging viral pathogens including dengue (DENV), West Nile (WNV) and Zika viruses (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout South East Asia, the Pacific islands, and the Far East, causing around 68,000 cases worldwide each year. There is no specific treatment for JEV. This study aims to determine the molecular structure of new potential drug targets for JEV. In this study, the JEV non-structural protein 1 C-terminal β-ladder domain (C-NS1) is presented at 2.1 Å resolution. The crystal structure of C-JEVNS1 shares a conserved fold with flavivirus C-NS1 domains. The surface charge distribution of C-JEVNS1 is similar to WNV and ZIKV but is significantly different from DENV. Analysis of the C-JEVNS1 structure, in silico molecular dynamics simulations and experimental solution small angle X-ray scattering, indicate extensive loop flexibility on the exterior of the protein. It is proposed that this together with charge distribution on the exterior of the protein influence NS1-host protein interaction specificity which may impact on pathogenicity. These factors may also affect the interaction with the monoclonal antibody, 22NS1, which is protective against WNV infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 participates in the interaction with lipidic membranes and that sulphate binding sites are not the glycosaminoglycans binding interfaces. For the first time, the crystal structure of the JEV capsid protein at 1.98 Å is also reported and compared to the existing flavivirus capsid protein. JEV capsid shows helical secondary structure (α helixes 1-4) and protein folding similar to DENV and WNV capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘), α helix 1-1’, 2-2’, and 4-4’. The capsid dimer is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix 1 of the capsid could be important for its function. This dimer model agrees with a previous suggestion that the capsid protein interacts with RNA via the basic rich C-terminal, α4-α4’, and associates with lipid bilayers at the opposite hydrophobic, α2-α2’.
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Grummitt, Charles Gordon. "The discovery and characterisation of the C-terminal domain of nucleophosmin : implications for Acute Myeloid Leukaemia with normal karyotype." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612508.
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Cho, Chi-kong Lawrence, and 曹智剛. "Structural characterization of C-terminal zinc finger domain of XIAP associated factor 1 (XAF1) and its interaction studies with XIAP." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47044123.
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Keyser, Rowena J. "Identifying ligands of the C-terminal domain of cardiac expressed connexin 40 and assessing its involvement in cardiac conduction disease." Thesis, Link to online version, 2007. http://hdl.handle.net/10019/651.
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Joo, Sang Hoon. "Synthesis and screening of support-bound combinatorial cyclic peptide and free C-terminal peptide libraries." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1195561420.
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Marston, Farhat Yasmeen. "NMR structure analysis and identification of the DNA binding site of the C-terminal domain of the Bacillus subtilis protein DnaD." Thesis, University of Sheffield, 2011. http://etheses.whiterose.ac.uk/14695/.
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The B. subtilis protein DnaD is an essential component of the chromosome replication machinery and a global regulator of DNA architecture, as it exhibits a unique DNA remodeling activity that opens up supercoiled DNA by forming higher order nucleoprotein structures. There are no homologues of this protein in gram negative bacteria and details of its molecular structure are at present limited to the crystal structures of the N-terminal domain from B. subtilis and G. Kaustophilus, and also crystal structures of the C-terminal domain of two proteins with unknown function from S. mutans and E. faecalis (Structural Genomics projects pdb codes 2zc2 and 2i5u respectively). In this thesis, the determination of the NMR structure for the DNA binding C-terminal domain of the B. sllbtilis DnaD protein is reported. This domain is composed of five helices and an unstructured C-terminal tail, helix I-IV form a well packed hydrophobic core and helix V, which is more extensive than assumed from sequence alignments, extends away from this core fold of the protein. NMR DNA titrations, in vitro mutagenesis and in vivo complementation experiments show that a highly conserved YxxxIxxxW motif, the final helix and a portion of the mobile Cterminal region make up the DNA binding module of B. subtilis DnaD and are all essential for cell viability. Sequence alignments of DnaD alone fail to identify two key elements of this DNA binding module, the poorly conserved sequences of the final helix and the C-terminal mobile segment. Sensitive Hidden Markov Models based techniques, show that the two structural domains found in DnaD are present in B. subtilis DnaB, a primosomal protein that also participates in replication initiation. Furthermore, these two proteins share the only strong sequence motif, the highly conserved YxxxIxxxW sequence that contributes to DNA binding.
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Schreieck, Amelie. "Role of the RNA polymerase II C-terminal domain in transcription termination and function of Spt5 in 3' RNA-processing factor recruitment." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-164544.
Full textAbstract:
Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II). This process is tightly regulated and coupled to RNA processing. Many transcription and RNA processing factors are recruited to Pol II via its conserved C-terminal domain (CTD) containing 27 heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 in Saccharomyces cerevisiae. These repeats can be differentially phosphorylated during the transcription cycle serving as a code for interacting factors. During transcription initiation, Ser5 is phosphorylated at the 5’-end of genes and this phosphorylation is required for RNA capping enzyme binding. During transcription elongation, the Pol II CTD becomes phosphorylated at Tyr1 and Ser2 and binds the elongation factor Spt5. Spt5 also contains a repetitive C-terminal region (CTR) required for cotranscriptional recruitment of proteins. At the 3’-end of genes, Ser2-phosphorylated Pol II associates with the cleavage and polyadenylation factor (CPF) and is dephosphorylated at Tyr1 residues.
This work shows that CPF is a Pol II CTD phosphatase and that its subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, Glc7 activity is required for normal Tyr1 dephosphorylation at the polyadenylation (pA) site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing and transcription termination are coupled via CPF-dependent Pol II Tyr1 dephosphorylation. Additionally, 19 kinases were tested for activity on Tyr1 in yeast by selective inhibition or knock-out in vivo. However, none of the candidates was identified as the Tyr1 kinase. Possibly this enzyme is an atypical kinase not known to be involved in transcription so far.
Furthermore, this work reports a new role of the Spt5 CTR in recruitment of RNA 3’-end processing factors. The results show that the Spt5 CTR as well as RNA is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3’-end of yeast genes. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 base pairs downstream of the pA site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at Ser2 of the CTD. Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo.
In summary, we characterized two new aspects of transcription and RNA processing regulation by two different C-terminal repetitive protein domains. CTD Tyr1 phosphorylation, which is removed by Glc7, regulates termination factor recruitment by masking their binding site, the Spt5 CTR is involved in recruitment of CFI. Both results greatly contribute to a more detailed understanding of the mechanisms involved in transcription termination and RNA
3’-end processing.
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Reddy, Edamakanti Chandrakanth [Verfasser], and Michael [Gutachter] Sendter. "Role of differential phosphorylation of c-Jun N-terminal domain in degenerative and inflammatory pathways of CNS / Edamakanti Chandrakanth Reddy. Gutachter: Michael Sendter." Würzburg : Universität Würzburg, 2013. http://d-nb.info/1103160605/34.
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Delaforge, Elise. "Dynamique structurale et fonctionnelle du domaine C-terminal de la protéine PB2 du virus de la grippe A." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV037/document.
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La capacité du virus de la grippe aviaire à traverser la barrière des espèces et à devenir fortement pathogène chez les mammifères est un problème majeur de santé publique. Chez les oiseaux, la réplication a lieu dans l'intestin, à 4C, tandis que chez les humains elle a lieu dans l'appareil respiratoire, plus froid, à 33C. Il a été montré que l'adaptation à la température du virus de la grippe a lieu par de nombreuses mutations de la polymérase virale, notamment dans le domaine 627-NLS situé en C-terminal de la protéine PB2. Ce domaine est impliqué dans l'adaptation à l'hôte et interagit avec la protéine de l'hôte, importine alpha, étant donc indispensable pour l'entrée de la polymérase virale dans le noyau de la cellule [Tarendeau et al., 2008]. Les structures cristallographiques du 627-NLS et du complexe importine alpha/NLS existent. Cependant, lors de la superposition de ces structures via leur domaine NLS commun, un important choc stérique entre le domaine 627 et l'importine alpha devient évident. Ceci indique qu'une autre conformation du 627-NLS est requise pour l'interaction avec l'importine alpha [Boivin and Hart, 2011]. Dans cette étude, nous avons examiné les bases moléculaires de l'adaptation inter-espèces du virus à travers l'étude de la structure et de la dynamique du 627-NLS aviaire et humain. Nous avons identifié deux conformations du 627-NLS en échange lent (10-100 s-1), correspondant apparemment à une conformation ouverte et une conformation fermée des deux domaines. Nous proposons que la conformation ouverte du 627-NLS est la seule conformation compatible avec l'interaction avec l'importine alpha, et que l'équilibre entre conformation ouverte et fermée pourrait jouer le rôle de thermostat moléculaire, contrôlant l'efficacité de la réplication virale chez différents hôtes. La cinétique et la dynamique de ce comportement conformationnel important ainsi que de l'interaction entre le 627-NLS et l'importine alpha ont été caractérisées par résonance magnétique nucléaire (déplacements chimique, augmentation paramagnétique de la relaxation, relaxation de spin, transfert de saturation par l'échange chimique), combinée à la diffusion des rayons X et des neutrons aux petits angles ainsi qu'au transfert d'énergie par résonance de type Förster. Aussi, nous avons déterminé les affinités d'une série de mutants évolutifs du 627-NLS pour l'importine alpha et du 627-NLS aviaire ou humain pour différents isoformes de l'importine alpha, montrant que les affinités observées sont cohérentes avec les préférences d'interactions vues in vivoThe ability of avian influenza viruses to cross the species barrier and become dangerously pathogenic to mammalian hosts represents a major threat for human health. In birds the viral replication is carried out in the intestine at 40°C, while in humans it occurs in the cooler respiratory tract at 33°C. It has been shown that temperature adaption of the influenza virus occurs through numerous mutations in the viral polymerase, in particular in the C-terminal domain 627-NLS of the PB2 protein. This domain has already been shown to participate in host adaptation and is involved in importin alpha binding and therefore is required for entry of the viral polymerase into the nucleus [Tarendeau et al., 2008]. Crystallographic structures are available for 627-NLS and the complex importin alpha/NLS, however, a steric clash between importin alpha and the 627 domain becomes apparent when superimposing the NLS domain of the two structures, indicating that another conformation of 627-NLS is required for binding to importin alpha [Boivin and Hart, 2011]. Here we investigate the molecular basis of inter-species adaptation by studying the structure and dynamics of human and avian 627-NLS. We have identified two conformations of 627-NLS in slow exchange (10-100 s-1), corresponding to an apparently open and closed conformation of the two domains. We show that the equilibrium between closed and open conformations is strongly temperature dependent. We propose that the open conformation of 627-NLS is the only conformation compatible with binding to importin alpha and that the equilibrium between closed and open conformations may play a role as a molecular thermostat, controlling the efficiency of viral replication in the different species. The kinetics and domain dynamics of this important conformational behaviour and of the interaction between 627-NLS and importin alpha have been characterized using nuclear magnetic resonance chemical shifts, paramagnetic relaxation enhancement, spin relaxation and chemical exchange saturation transfer, in combination with X-ray and neutron small angle scattering and Förster resonance energy transfer. Also, we have determined the affinities of various evolutionnary mutants of 627-NLS to importin alpha and of avian and human 627-NLS to different isoforms of importin alpha, showing that the observed affinities are coherent with the preferred interactions seen in vivo
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Chirgadze, Dmitry Yurievich. "Crystallographic studies of signalling proteins : amino-terminal domain of protein kinase C-related kinase 1 and NK1 fragment of hepatocyte growth factor/scatter factor." Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312646.
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Kuznetsova, Olga. "Regulation of human RNA polymerase II CTD modifications." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:e745b5b6-8a4a-4b7a-81d7-499bca8bfea1.
Full textAbstract:
Transcription of human protein-coding genes and most small nuclear RNA genes is mediated by RNA Polymerase II (Pol II). During a cycle of transcription, Pol II recruits a variety of factors that facilitate transcription elongation, RNA processing and termination, through its long, unstructured C-terminal domain (CTD). The CTD in humans comprises 52 tandem heptapeptide repeats with the consensus sequence Y1S2P3T4S5P6S7. Each amino acid of the heptapeptide can be chemically modified, which influences the recruitment of other protein factors to the transcription machinery. Not all enzymes that modify the CTD have been discovered. Recent studies have identified a novel CTD phosphatase: RPAP2 in humans and its yeast homologue Rtr1, which dephosphorylate phospho-Ser5 of the heptapeptide repeats. RPAP2 has been shown to stimulate 3' end cleavage of nascent snRNAs through recruitment of the Integrator complex, and unpublished work suggests the involvement of RPAP2 in regulating vertebrate developmental programs. However, the exact mechanisms that regulate the function of human RPAP2, and thus impact on CTD modification, are not well-understood. This thesis presents a novel mechanism whereby RPAP2 recruits protein phosphatase 1 (PP1) to snRNA genes, where PP1 is postulated to activate P-TEFb to phosphorylate Ser2 of the CTD. At the same time, P-TEFb may have a role in activating the phosphatase activity of RPAP2. Furthermore, RPAP2 itself is shown to be recruited to a number of gene promoters by the RPRD1A protein, which also stimulates its phosphatase activity. RPAP2 was shown to have another role in regulating transcription termination: by recruiting the Integrator complex, which is shown here to mediate termination of snRNA genes, and by a so far unknown mechanism on a long protein-coding gene. An attempt was made to purify and crystallise the human RPAP2 to obtain a crystal structure, however the crystallisation trials were not successful. Finally, a correlation was found in human embryonic stem cells and induced pluripotent stem cells between low levels of RPAP2 and high levels of CTD Ser5P, suggesting a potential involvement of RPAP2 in regulating transcription at a key developmental stage. The results presented here contribute to the understanding of human transcriptional mechanisms and the numerous interactions within the transcription machinery. In particular, the mechanism of terminating the transcription of snRNA genes is identified. An interesting possibility is the regulation of development and stem cell differentiation by RPAP2; however the exact pathways by which this occurs are yet to be discovered.
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Finney, Angela H. "Role of the C-terminal domain of the a subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/36285.
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Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, Vibrio fischeri. In V. fischeri, the luminescence or lux genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the lux operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the aCTD of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effect of seventy alanine substitution variants of the a subunit was determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by in vitro assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxRDN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDN, and fourteen residues in the aCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxRDN-dependent activation in vivo and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type aCTD is capable of interacting with the lux DNA fragment tested, all five of the variant forms of the aCTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that aCTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the lux operon during quorum sensing.Master of Science
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Rabara, Taylor Renee. "Study of Physical Protein-Protein Interactions Between the MaSp1 C-Terminal Domain and Small Cysteine-Rich Proteins Found in the Major Ampullate Gland of Latrodectus hesperus." Scholarly Commons, 2016. https://scholarlycommons.pacific.edu/uop_etds/2965.
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Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies
which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation.
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Ellena, Rachel A. "Antimicrobial and lipid binding properties of the C-terminal domain of apolipoprotein A-I determined using a novel apolipophorin III/apolipoprotein A-I (179-243) chimera." Thesis, California State University, Long Beach, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10144827.
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Apolipoprotein A-I (apoA-I) is an exchangeable apolipoprotein that constitutes the major protein component of high density cholesterol. ApoA-I is a two-domain protein comprising an N-terminal helix bundle and a less-structured C-terminal domain in the lipid-free state. In the present study, the contribution of the C-terminal domain to the lipid binding and antimicrobial activity of apoA-I was investigated using a chimeric construct in which the C-terminal domain of apoA-I (179-243) was attached to an insect apolipoprotein, Locusta migratoria apolipophorin III (apoLp-III), bearing cysteine substitutions for residues 20 and 149. Circular dichroism results were consistent with the addition of a poorly structured domain to apoLp-III and revealed the apoLp-III helix bundle was successfully closed under oxidizing conditions. Electrophoresis, fluorescence spectroscopy and an in vitro study using macrophage cells revealed that the C-terminal domain in itself was insufficient for efficient binding to lipid, lipopolysaccharide and phosphatidylglycerol vesicles. These results suggest the underlying mechanisms governing these interactions are potentiated by cooperativity between the N- and C-terminal domains of apoA-I.
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Schreieck, Amelie [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Role of the RNA polymerase II C-terminal domain in transcription termination and function of Spt5 in 3' RNA-processing factor recruitment / Amelie Schreieck. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1046503308/34.
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FAGGION, Beniamino. "Expression and structural studies of insulin-like growth factor binding proteins and the c-terminal domain of perlecan." Doctoral thesis, 2007. http://hdl.handle.net/11562/337974.
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Insulin-like Growth Factors (IGFs), Insulin-like Growth Factor Binding Proteins
(IGFBPs) e Insulin-like Growth Factor Binding Proteins-related Proteins (IGFBP-rP).
Le IGF-I e IGF-II sono piccoli ormoni peptidici di 70 e 67 amminoacidi
rispettivamente. Essi sono così chiamati a causa della loro alta omologia con
l’insulina. Le IGF hanno effetti a livello cellulare promuovendo la crescita e il
differenziamento, vengono legate in vivo dalle IGFBP e da 2 tipi di recettori
(recettore tipo 1 e tipo 2) che mediano i loro effetti. Le IGFBP costituiscono una
famiglia di 6 proteine (da IGFBP-1 a -6) le cui dimensioni vanno da 216 a 289
amminoacidi di lunghezza. Dal punto di vista strutturale, esse sono suddivise in
tre domini: un dominio ammino-terminale conservato ricco in cisteine, una
regione centrale non conservata e un dominio carbossi-terminale conservato. La
capacità di legare IGF permette alle IGFBP di regolare la concentrazione di IGF
circolanti, di sequestrarle o di concentrarle in prossimità dei tessuti bersaglio. Le
IGFBP però possono dare degli effetti biologici anche indipendentemente dal
legame con le IGF. E’ stato dimostrato, ad esempio, da studi condotti utilizzando
frammenti delle IGFBP che questi sono in grado di influenzare l’adesione cellulare
attraverso il legame con le integrine. Nel complesso, il sistema delle IGF è
coinvolto in numerosi processi nell’organismo e sempre più patologie sono state
correlate al suo malfunzionamento.
Successivamente, sono state identificate altre proteine che mostrano omologia
per le IGFBP, limitatamente al dominio ammino-terminale, le quali possono legare
le IGF con affinità inferiore rispetto alle IGFBP. Si è proposto quindi di chiamare
queste proteine IGFBP-related proteins.
In questo lavoro di tesi la IGF-II umana ricombinante è stata espressa in
Escherichia coli , inizialmente come proteina depositata in corpi d’inclusione e
successivamente in forma solubile. La proteina prodotta in corpi d’inclusione è
stata solubilizzata, purificata mediante cromatografia di affinità (tramite una coda
di 6 istidine legata alla proteina), ed è stata intrapresa la rinaturazione. La
proteina così ottenuta è stata cristallizzata ma i cristalli ottenuti non hanno
consentito di determinarne la struttura mediante analisi di diffrazione di raggi X.
La proteina in forma solubile è stata ottenuta cambiando vettore di espressione
ma la bassa specificità per la matrice di affinità usata ha posto problemi per la
sua purificazione.
E’ stata poi espressa la IGFBP-3 umana utilizzando il lievito metilotrofico Pichia
pastoris . La proteina è stata purificata mediante cromatografia a scambio ionico e
gel-filtrazione ed è stata tentata la cristallizzazione che però non ha dato
risultati.
Inoltre è stata espressa e purificata la IGFBP-rP1 in Escherichia coli . La proteina è
stata purificata mediante cromatografia di affinità (utilizzando una resina Nickel
Sepharose che lega la coda di istidine fusa alla proteina ricombinante), e
utilizzata per l’allestimento delle prove di cristallizzazione preliminari. Una delle
condizioni testate ha dato dei cristalli che però non possono ancora essere usati
per risolvere la struttura tridimensionale.
Dominio LG-3 (Laminin G-like) del perlecano umano.
Il perlecano umano è un eparan-solfato proteoglicano, costituito da un nucleo
proteico del peso di 470 kDa legato a oligosaccaridi e catene di eparan solfato
che portano il peso complessivo del perlecano fino a 800 kDa. Esso costituisce,
insieme a collagene e laminine la membrana basale, cioè una matrice
specializzata presente nell’endotelio, nell’epitelio e attorno alle cellule muscolari.
Il dominio carbossi-terminale del perlecano (dominio V del peso di 85 kDa) è
chiamato endorepellina ed è stata dimostrata la sua capacità di funzionare come
fattore anti-angiogenico.
Il dominio LG-3 del perlecano umano è stato espresso come proteina
ricombinante in Escherichia coli e purificato tramite cromatografia di affinità
sfruttando una coda di istidine. La proteina è stata quindi cristallizzata ed è stata
determinata la sua struttura tridimensionale mediante analisi di diffrazione di
raggi X.
α1-Microglobulina.
L’α1-microglobulina è una lipocalina, è costituita da 183 amminoacidi e pesa 26
kDa. L’α1-microglobulina umana è glicosilata in tre posizioni, due delle quali
sono siti di N-glicosilazione e una e un sito di O-glicosilazione. Essa viene
sintetizzata nel fegato come proteina di fusione insieme ad un’altra proteina, la
bikunina, e viene successivamente tagliata e immessa nel plasma. Una importante
funzione della α1-microglobulina è la sua capacità di modulare negativamente
l’attività del sistema immunitario proteggendo i tessuti dalla risposta
immunitaria.
L’ α1-microglobulina di topo è stata espressa come proteina ricombinante
utilizzando il lievito Pichia pastoris . La proteina è stata purificata tramite
cromatografia a scambio ionico e gel filtrazione. La proteina purificata è stata
quindi utilizzata per l’allestimento delle prove preliminari di cristallizzazione che
però non hanno dato risultati.Insulin-like Growth Factors (IGFs), Insulin-like Growth Factor Binding Proteins (IGFBPs) and Insulin-like Growth Factor Binding Proteins-related Proteins (IGFBPrP). IGF-I and IGF-II are small hormone peptides of 70 and 67 amino acids respectively and are called insulin-like because of their homology with insulin. IGFs promote cellular growth and differentiation, bind IGFBPs in vivo and also two types of receptors (type 1 and type 2 receptor) which mediate their actions. The IGFBPs form a six member family (IGFBP-1 to -6) and are between 216 to 289 amino acids long. They are subdivided into three distinct domains: a conserved amino-terminal cysteine-rich region, a non-conserved midregion, and a conserved carboxy-terminal region. The IGFBPs bind the circulating IGFs thus regulating their concentration, by sequestering them or releasing them in proximity of their target tissues. In addition, IGFBP effects independent of IGFbinding have been demonstrated. Studies with proteolyzed fragments of IGFBPs unable to bind IGFs, have shown their ability to influence cellular adhesion by integrin binding. The IGF system is involved in various processes and many pathological events have been related to its failure. In addition, other proteins have been identified which have the ability to bind IGFs with lower affinity, compared to IGFBPs. These proteins show homology with IGFBPs in their amino-terminal region and are called IGFBP-related proteins. During this thesis, human IGF-II was expressed in Escherichia coli , in the form of inclusion bodies and in its soluble form. The protein from inclusion bodies was solubilized, purified by affinity chromatography (using a 6 histidine tag), and then refolded. The renatured protein was crystallized but with the data obtained from those crystals it was not possible to determine the three dimensional structure. The soluble form was obtained by using a different expression vector and a 6 histidine tag but purification using the tag was not possible due to the low affinity for the chromatography resin. Human IGFBP-3 was also expressed using the methylotrophic yeast Pichia pastoris . The recombinant protein was purified by ion-exchange chromatography and by gel-filtration but the crystallization trials did not yield good crystals. Human IGFBP-rP1 was expressed in Escherichia coli , purified by affinity chromatography and used for preliminary crystallization trials. One crystallization condition gave small crystals which are still not suitable for X-ray diffraction analysis. LG-3 (Laminin G like) domain of the human perlecan Human perlecan is an heparan-sulphate proteoglycan, containing a protein core of 470 kDa with oligosaccharides and heparan sulphate chains attached to it which give a total molecular weight of 800 kDa. Together with collagen and laminin it forms the basement membrane which is a specialized matrix found in epithelium, endothelium and surrounding muscle cells. The carboxy-terminal domain of perlecan (domain V of 85 kDa) is called endorepellin and possesses anti-angiogenic activity The LG-3 domain of human perlecan was expressed in Escherichia coli and purified by affinity chromatography. The purified protein was then crystallized and its molecular structure was determined by X-ray diffraction analysis. α1-Microglobulin. α1-Microglobulin is a lipocalin of 26 kDa and is 183 amino acids long. Human α1-microglobulin is glycosylated in three positions: two of these are of the Nlinked type and the third one is of the O-linked type. α1-Microglobulin is synthesized in the liver as a fusion protein with bikunin from which it is subsequently cleaved and released into plasma. The biological function of α1- microglobulin is related to its ability to negatively modulate the immune system activity thus preventing tissue damage by immunological responses. Mouse α1-microglobulin was expressed as a recombinant protein by using the yeast Pichia pastoris . The protein was purified by ion-exchange chromatography and gel filtration and preliminary crystallization trials were prepared which gave no crystals.
47
Chang, Shiao-Ru, and 張筱茹. "Functional study of C-terminal domain on KAP1." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/46251599936660592030.
Full textAbstract:
碩士國立中興大學
分子生物學研究所
98
Transcription regulation plays a critical role in eukaryotic gene expression. KAP1 (KRAB-associated protein) is a transcriptional intermediary factor for repression, which asts as scaffold in many transcriptional regulation complexes. KAP1 serves as a co-repressor for KRAB-containing zinc finger proteins (KRAB-ZFPs) in the regulation of gene expression and KAP1 is required for KRAB-ZFPs-mediated transcription repression. However, the molecular mechanism of KAP1 functions as a co-repressor to connect transcription factors and regulatory proteins is unclear. Previous studies proved that N-terminal RBCC domain of KAP1 is responsible for oligomerization and KAP1 exists as both oligomers and monomers in cells, but how KAP1 oligomerization regulates transcriptional repression remains unknown. First, we want to understand wether KAP1 oligomerization affects its function on transcriptional repression. In addition, it has been previously demonstrated that C-terminal bromodomain (BD) of KAP1 regulates subcellular localization and transcriptional activity by interacting with its N-terminal RBCC domain. Above studies suggest that intramolecular and intermolecular interaction of KAP1 might cooperative regulate subcellular localization and transcriptional activity of KAP1. In this study, we want to address the molecular mechanism of intermolecular and intramolecular interaction of KAP1 regulates its subcellular localization and transcriptional activity. To investigate the role of KAP1 oligomerization in transcriptional repression, we blocked the self-interaction of KAP1 to mimic monomeric KAP1 by generating point mutations on RBCC domain. Here we demonstrated that monomeric KAP1 exhibited a decreased level of euchromatin localization but stronger repressional activity. The results suggest that monomeric KAP1 is an active form for transcriptional repression. Go further to explore the mechanism of KAP1 oligomerization in subcellular localization and transcriptional activity, we approach it from C-terminal domain of KAP1. Previously we found that missing of BD in KAP1 results in spots formation in euchromatin region and derepressional activity, but these phenomena could be reverted to wild type by addition of BD which through intramolecular interaction with RBCC domain. Thus, we proposed that intramolecular interaction mediated by BD of KAP1 contribute to subcellular localiztion and transcriptional activity by promoting KAP1 monomers formation to repress transcription, which blocks the RBCC domain for KAP1 oligomerization. To prove this idea, first, we perform co-immunoprecipitation assay to prove C-terminal BD interacts with N-terminal RBCC domain. To validate that BD of KAP1 regulates its oligomerization by intramolecular interaction, we demonstrated that missing BD of KAP1 leads to oligomers formationin glycerol gradient analysis, which reverted to monomers by addition of BD. Furthermore, we demonstrated that BD interacts with RBCC domain of KAP1, suggesting that intramolecular interaction mediated by BD regulates KAP1 oligomerization. Taken together, the results demonstrate that intramolecular interaction between C-terminal BD and N-terminal RBCC domain regulates KAP1 monomers formation, which is capable of heterochromatin localization and transcriptional repression.
48
Dias, João Rodrigo Diogo. "Investigating lysine methylation of RNA polymerase II C - terminal domain." Doctoral thesis, 2016. https://repositorio-aberto.up.pt/handle/10216/88575.
Full text
49
Dias, João Rodrigo Diogo. "Investigating lysine methylation of RNA polymerase II C - terminal domain." Tese, 2016. https://repositorio-aberto.up.pt/handle/10216/88575.
Full text
50
Chen, Hung-Mei, and 陳宏美. "Construction,Expression and Purification of the N-terminal and C-terminal domain of the ApoE." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/98593584575260222495.
Full textAbstract:
碩士國立中正大學
分子生物研究所
92
Apolipoprotein E (ApoE) is a plasma apolipoprotein of 299 amino acids (Mr 34,000 ) that is primarily synthesized in livers. ApoE plays an important role in lipoprotein transport and cholesterol clearance. ApoE is also produced and secreted in the brain and is believed to participate in neuronal regeneration. Three ApoE isoforms, i.e. ApoE2, ApoE3, and ApoE4, have been identified in human being. The most common isoform is ApoE3. A higher prevalence of ApoE2 was observed in hypertriglyceridemic patients, while ApoE4 was noted in hypercholesterolemic patients. ApoE has been proven to influence its metabolic properties. Recent studies have revealed that ApoE interacts with the senile plaque in the brains of Alzheimer disease. The frequency of ApoE4 isoform is significantly higher in the population of late-onset Alzheimer disease. The previous studies have shown that ApoE contains two structural domains, the NH2-terminal and the COOH-terminal domains. The NH2-terminal domain contains the lipoprotein receptor binding region and consists of a four-helix bundle with antiparallel arrangement. The COOH-terminal domain contains the lipoprotein (lipid) binding site. The structure of the COOH-terminal domain of ApoE, however, is unknown, but is supposed to be highly amphipathic α-helices. The interaction between N-terminal and C-terminal domains of ApoE has been proven to influence its biochemical properties. The purpose of this study is aimed to express and purify a large amount of ApoE COOH-terminal domain in E.coli through non labeled or labeled-medium followed by investigating its structure by NMR studies.