Rozprawy doktorskie: „Domain Studies”

1

Batra, Sharat. "Magneto-optical studies of domain wall oscillations /". The Ohio State University, 1986. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487265555439935.

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2

Parnas, Henrietta. "Binding studies of the SH3D domain of intersectin". Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=87005.

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The scaffolding protein intersectin1 is involved in clathrin-mediated endocytosis, apoptosis, signal transduction and regulation of cytoskeletal rearrangements. Thus, understanding the interactions of intersectin1 with other proteins has important consequences for cell biology. It had previously been shown that Cdc42 GTPase-activating protein (CdGAP) is inhibited by intersectin1, resulting in increased actin polymerization and lamellipodia formation. The central basic-rich region of CdGAP has been shown to bind to the intersectin1 SH3D, despite not containing any previously described SH3 binding motifs. Several CdGAP peptides from its basic-rich region were found to bind to the intersectin1 SH3D in peptide array experiments, and therefore were proposed to contain novel SH3 binding motifs. Binding of a peptide causes specific chemical shift perturbations in the nuclear magnetic resonance (NMR) spectrum of a protein. In this study NMR spectroscopy was used to validate the interaction of the intersectin1 SH3D domain with peptides from several putative binding partners. The basic-rich CdGAP peptides identified in the peptide array experiments did not bind to recombinant intersectin1 SH3D in NMR titrations. Two peptides from dynamin containing typical SH3 binding motifs did not bind to intersectin1 SH3D; however, a longer proline-rich peptide from synaptojanin binds weakly. Based on homology to this synaptojanin peptide, a novel CdGAP peptide was identified that did bind to the intersectin1 SH3D. Chemical shift data was used to map the binding sites of each peptide onto the previously published structure of intersectin2 SH3D. The binding sites of the two peptides overlap, and include residues that are highly conserved amongst SH3 domains. This is the first identification of a peptide from CdGAP that binds to intersectin1 SH3D, and its homology to the synaptojanin peptide helps define the atypical binding motif of the intersectin1 SH3D domain. This study se
La protéine structurale intersectine1 est impliquée dans l'endocytose, l'apoptose, la transduction de signaux ainsi que la régulation des réarrangements du cytosquelette. La compréhension des interactions d'intersectine1 avec d'autres protéines a des conséquences importantes pour la biologie cellulaire. L'activité de la protéine activatrice de la GTPase Cdc42 (CdGAP) est empêchée par l'intersectine1 ce qui augmente la polymérisation des d'actine. La région centrale basique de CdGAP qui contrôle la liaison de la protéine avec ses substrats ne contient aucun motif qui se lie aux domaines SH3. Quelques peptides de de cette région se lient à intersectine1 SH3D en utilisant un criblage d'une banque de peptides, et ont été proposés de contenir des motifs SH3 nouveaux. La liaison avec un peptide entraîne des perturbations de déplacements chimiques dans le spectre de résonance magnétique nucléaire (RMN) d'une protéine. Dans cette recherche, la RMN fut utilisée pour étudier l'interaction du domaine SH3D de l'intersectine1 avec des peptides de quelques associés putatifs. Les peptides de CdGAP qui furent identifiés lors du criblage d'une banque de peptides ne se lient pas à l'intersectine SH3D recombinante. Deux peptides de la protéine dynamine qui contiennent des motifs SH3 typiques ne se lient pas avec l'intersectine1 SH3D, mais un plus long peptide de synaptojanine, riche en proline, se lie faiblement. Un peptide de CdGAP, identifiée par homologie avec ce peptide, s'est aussi lié. Les changements de déplacements chimiques furent utilisées pour déterminer le site de liaison sur la structure de l'intersectine2 SH3D. Les sites de liaison des deux peptides se superposent. Cette liaison se produit avec des acides aminés qui sont conservés parmi les domaines SH3. Ceci est le premier peptide de CdGAP qui se lie à l'intersectine1 SH3D. Son homologie au peptide de synaptojanine peut aider la caractérisation de ce motif de liai

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3

Menderin, Nathan. "Studies on the Human Sp1 DNA-Binding Domain". Thesis, University of Exeter, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507135.

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4

Hardman, C. H. "NMR studies of the A domain of HMG1". Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603702.

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This thesis describes the solution structure of the A domain of rat HMG1. First, the A domain from rat HMG1 was subcloned, over-expressed and purified from E. coli. Initial characterisation revealed a mixed population of protein species resulting from both intra- and inter-molecular disulphide bond formation. To produce a suitable sample for NMR spectroscopy the non-conserved cysteine 22 was replaced with serine by site-directed mutagenesis. This mutant protein resembles the wild-type in binding to four-way junctions and short DNA duplexes. CD and NMR spectra suggest no major structural perturbations, and the protein remains fully reduced after many weeks in solution. The structure is very similar to that of the B domain. This together with the structure of Drosophila HMG-D protein (solved during this study), confirms that different HMG-boxes from the superfamily are likely to have the same global fold. Comparison with the recent structures of two different sequence-specific HMG-box:DNA complexes reveals both some similarities and differences. Some of the differences between the A domain and the other HMG structures were confirmed by studies of backbone dynamics of the protein (studied by others). The structure provides a framework to interpret many of the mutations generated in the HMG-box, and the starting point to discover the molecular differences responsible for the different properties of A and B boxes of HMG1 and between sequence and non-sequence specific HMG-box domains. The availability of isotopically labelled protein with full ¹H, ¹³C and ¹⁵N assignments and known three-dimensional structure will provide the means to examine directly the nature of structure-specific DNA recognition by this novel class of protein using NMR spectroscopy.

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5

Edwards, Simon David. "Structural studies of the FERM domain of moesin". Thesis, Birkbeck (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268755.

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6

Berthoumieu, Olivia. "Single molecule studies of seven transmembrane domain proteins". Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:ff7ae71d-5481-4523-812b-2128fe32f5fc.

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This work aimed at studying biophysical properties of two membrane proteins, one of potential nanotechnological use, bacteriorhodopsin, and one potential drug target, the NTS1 neurotensin receptor, at the single molecule scale. Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) of the halophilic organism Halobacterium salinarium. It is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of its retinal chromophore. Its stability, as well as its photoactivity remaining in dry protein layers, has made BR an attractive material for biomolecular devices. Numerous studies have been published on this topic; however, they have all used BR within the PM, on relatively large (µm-wide) surfaces. Here, conducting-probe atomic force microscopy (C-AFM) analysis was performed after removing most of the membrane lipids. For the first time, it was shown that the molecular conductance of BR can be reversibly photoswitched with predictable wavelength sensitivity. Intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine which, combined with partial delipidation, generated protein trimers homogenously orientated on the surface. Numerous controls using biophysical (SPR, ellipsometry, Kelvin-probe AFM) and chemical (photocurrent, cyclic voltammetry) techniques confirmed the wavelength specificity of the photoswitch, the anchoring role of the mutation and the homogenous orientation of the protein on the gold surface. Neurotensin is a brain and gastrointestinal 13 amino acid peptide acting as a neuromodulator in the central nervous system and as a hormone in the periphery. Its wide range of biological activities is primarily mediated through its binding to the neurotensin type 1 receptor (NTS1). NTS1 expressed in E.coli was purified and inserted into 100 nm brain polar lipid liposomes in a conformation which retained its ligand-binding capabilities. Initial AFM characterisation was performed as a prelude for ligand-receptor interaction studies, including high resolution imaging, force spectroscopy and solid state NMR approaches.

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7

Williams, Samantha Catherine. "Studies of the domain structure of complement factor B". Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358907.

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8

Ali, M. "Structural studies of Xenopus laevis Quaking protein QUA1 domain". Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595447.

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A recent report showed that a specific point mutation of glutamate to glycine (E48G) in the QUA1 domain affects both the oligomerisation and the RNA-binding properties of the murine Quaking homologue (QkI). The mutation has also been shown to be embryonic lethal in mice at around day 10 of gestation. The aims of this thesis were to investigate the structural features of QUA1 domain and explain why E48G mutants fail to dimerize. The first objective was pursued by expression and purification of the Xenopus laevis Quaking homologue (pXqua) QUA1 motif, followed by biophysical characterization and structural studies using solution-state nuclear magnetic resonance (NMR) spectroscopy. The second objective was addressed by biophysical characterization of an E48G mutant of the QUA1 domain, prepared using site directed mutagenesis. The QUA1 dimer was shown to comprise two α-helical hairpins assembling via an unusually small and polar protein-protein interface. Based on this structure, we proposed that the properties of the E48G mutant could be explained by disruption of salt-bridge interactions, either between E43 and R46 of the same protomer or R38 of the other protomer. This hypothesis was tested by generating a series of QUA1 mutants by site directed mutagenesis and analyzing them using analytical SEC, both as cleaved proteins and as MBP fusion proteins. Moreover, a separate mutational analysis was performed to compare the pXqua QUA1 domain with the corresponding regions in other STAR/GSG proteins. Sequence alignment of the QUA1 region showed proline residue in the predicted helix α1 of SAM68 and SF1. Corresponding mutations were made in pXqua QUA1 domain and the effect was investigated by studying their oligomerisation.

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9

Pommer, Ansgar J. "Mechanistic studies on the DNase domain of colicin E9". Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361425.

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10

Scott, Nerida Robyn. "Folding studies of the glucocorticoid receptor DNA-binding domain". Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627279.

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11

Thompson, Elizabeth Claire. "Studies on SET and MYND domain proteins in Drosophila". Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612456.

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12

Leung, Adelaine Kwun-Wai. "Structural studies of the spliceosomal U4 snRNP core domain". Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614942.

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13

Tran, Huong Jade Thien Thi. "Structural and functional studies of JAB1/MPN domain proteins". Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614104.

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14

Purves, Maud. "The D-domain of fibrin : structural and functional studies". Doctoral thesis, University of Cape Town, 1987. http://hdl.handle.net/11427/27202.

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The D-domain of fibrin (ogen) was separated from the parent molecule by plasmin digestion in the presence of calcium and isolated by means of DEAE-anion exchange chromatography followed by gel-filtration in buffer containing 4 M urea. Fluorescent-D-dimer (f-D-dimer) was isolated from a plasmic digest of fibrin clotted in the presence of 2.45 mM dansyl cadaverine, a fluorescent lysine analogue. Fluorescent-D-monomer was a by-product of f-D-dimer purification, the yield being determined by the concentration of dansyl cadaverine. At 2.45 mM f-D-monomer was always present in the digest. The f-D-monomer is probably formed directly and not as a result of degradation of f-D-dimer. The molecule elutes in the fibrinogen-derived-D- monomer position on gel-filtration. A protease was isolated and partially purified from venom of the puffadder (Bitis arietans). Puffadder venom protease is characterized by its ability to cleave D-dimer into symmetrical D-monomers, smaller than plasmin-derived D-monomers from fibrinogen. This characteristic was used to detect the puffadder venom protease activity with fluorescent-D-dimer being used as the substrate. Fractions obtained were assayed for D-dimer cleavage activity and the samples analyzed by means of SDS-PAGE on 4-20% gradient gels under reducing (βME) and non-reducing conditions. The fluorescent bands were located under U.V light and photographed prior to staining with Coomassie Blue. Several methods for the purification of the protease were investigated.

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15

Wu, Dimao. "Synthetic Studies on Phorboxazole Tail Domain and Amphidinolide C". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354560325.

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16

Schmiedeskamp, Mia Ruth. "NMR studies of the DNA-binding domain of ADR1 /". Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/9208.

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17

Patterson, C. J. F. "Topographic, ultrasonic and diffraction studies of helical antiferromagnets". Thesis, University of Hull, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381882.

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18

Powell, Ryan R. "Outback Nevada| Public Domain and Environmental Challenge". Thesis, University of Nevada, Reno, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10633860.

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With the arrival of Euro-Americans to Nevada, settlers and travelers experienced struggles and opportunities on Nevada’s marginal lands. These lands did not fit well with Euro-American ideas of progress and resource-use throughout the second part of the nineteenth century. After 1848, these marginal lands became part of America’s public domain with little promise for permanent settlements. Between 1860 and 1905, Euro-Americans imposed unsustainable land-uses on Nevada’s marginal lands. Due to increased competition on limited rangelands, federal land managers working for the United States Forest Service (USFS) came to Nevada after 1905 and secured the water resources in the highest mountains to promote favorable conditions of water flows for preferred local settlers. These settlers were the cattle ranchers with permanent home ranches that depended on water from the high mountains for summer grazing and haymaking. In the early twentieth century, beginning with the creation of the USFS in 1905 and ending with the Taylor Grazing Act in 1934, federal land managers were critical to maintaining successful settlements on a challenging environment in outback Nevada.

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19

Zhang, Min. "Crystallographic studies of the E. coli puta proline dehydrogenase domain /". free to MU campus, to others for purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p1426119.

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20

Walke, Stefan. "Biochemical and structural studies of the spliceosomal snRNP core domain". Thesis, University of Cambridge, 2000. https://www.repository.cam.ac.uk/handle/1810/251736.

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21

Patel, Pryank. "Structural studies of the inner DysF domain of human myoferlin". Thesis, Birkbeck (University of London), 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479383.

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22

Antoniadou, Eleni. "Studies on the biochemistry of the targetin domain of Lysostaphin". Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411404.

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23

Jones, Gareth. "NMR studies of the DNA-binding domain of B-Myb". Thesis, University of Kent, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270703.

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24

Meng, Ling Guang. "Frequency domain methods for turbogenerator and power system transient studies". Thesis, University of Newcastle Upon Tyne, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308982.

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25

Watson, Martin. "Structural studies on the PX domain of sorting nexin 1". Thesis, University of Sheffield, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434574.

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26

Joo, Chulmin 1976. "Spectral-domain optical coherence phase microscopy for quantitative biological studies". Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/43142.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.
Includes bibliographical references.
Conventional phase-contrast and differential interference contrast microscopy produce high contrast images of transparent specimens such as cells. However, they do not provide quantitative information or do not have enough sensitivity to detect nanometerlevel structural alterations. We have developed spectral-domain optical coherence phase microscopy (SD-OCPM) for highly sensitive quantitative phase imaging in 3D. This technique employs common-path spectral-domain optical coherence reflectometry to produce depth-resolved reflectance and quantitative phase images with high phase stability. The phase sensitivity of SD-OCPM was measured as nanometer-level for cellular specimens, demonstrating the capability for detecting small structural variation within the specimens. We applied SD-OCPM to the studies of intracellular dynamics in living cells and the detection of molecular interactions on activated surfaces as a sensor application.In the study of intracellular dynamics, we measured fluctuation of localized field-based dynamic light scattering within cellular specimens. With its high sensitivity to amplitude and phase fluctuations, SD-OCPM could observe the existence of two different regimes in intracellular dynamics. We also investigated the effect of an anti-cancer drug, Colchicine and ATP-depletion on the intracellular dynamics of human ovarian cancer cells, and observed the modification in diffusion characteristics inside the cells. Based on the optical sectioning capability of SD-OCPM, quantitative phase imaging was performed to examine slow dynamics of living cells. Through time-lapsed imaging and spectral analysis on the dynamics at the vicinity of cell membrane, we observed the existence of dynamic and independent sub-domains inside the cells that fluctuate at various dominant frequencies with different frequency contents and magnitudes.SD-OCPM was further utilized to measure molecular interactions on activated sensor surfaces.
(cont) The method is based on the fact that phase varies as analyte molecules bind to the immobilized probe molecules at the sensing surface and SD-OCPM can measure small phase alteration at any surface with high sensitivity. We have measured a -1.3 nm increase in optical thickness due to the binding of streptavidin on a biotin-activated substrate in a micro-fluidic device. Moreover, SD-OCPM was extended to image protein array chips, demonstrating its potential as a multiplexed protein array scanner.
by Chulmin Joo.
Ph.D.

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27

Suckling, Richard John. "Structural and functional studies of [delta]-COP Mu-homology domain". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607739.

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28

Wang, Wooi Koon. "Biophysical and functional studies of SAM Domain of human p73α". Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621141.

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29

Morgan, Hugh P. "Structural and biochemical studies of cold shock domain containing proteins". Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/15432.

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This thesis describes a novel DNA microarray method for determining the sequence specificity of single-stranded nucleic acid binding proteins (SNABPs). In Salmonella typhimurium six homologous CSPs (StCspA, StCspB, StCspC, StCspD, StCspE, StCspH) have been identified, although their functions are yet to be clearly elucidated. The novel microarray assay revealed two different types of ss DNA binding preferences for either purine or pyrimidine rich sequences. CspD bound purine (guanine) rich sequences, with the consensus binding sequence, 5’-ACGGgg-3’. CspA, CspB, CspC, and CspE bound pyrimidine (thymine) rich sequences, with an identical consensus core binding sequence. 5’-TCTTT-3’. The kinetics and thermodynamics of CSP/ss DNA interactions were examined for StCspE and StCspD, using the surface plasmon resonance method and isothermal titration calorimetry, which complemented the initial results determined by the novel microarray method. In addition, the X-ray crystal structure of StCspE was determined at 1.1 Å resolution and refined to R = 0.203. A computer generated model of StCspD was also created. The consensus ss DNA binding sequences for StCspE and StCspD (5’GTCTTTT-3’ and 5’-ACGGGG-3’, respectively), were docked onto the structures to reveal key molecular interactions, which accounted for the observed ss DNA sequence specificities. This work reveals key differences in selective ss DNA binding, existing within a small highly conserved family of CSPs, thus reflecting potential differences in function. Classification of SNABPs in this manner may provide a means of elucidating their cellular function and identifying gene networks regulated by specific SNABPs.

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30

Sekharan, Monica R. "Structural studies of the cGMP-binding GAF domain of PDE5A /". Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/8502.

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31

Ito, Yoshimichi. "Frequency Domain Studies on Sampled-Data Systems Using FR-Operators". 京都大学 (Kyoto University), 2011. http://hdl.handle.net/2433/142255.

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32

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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33

Nanao, Max H. "Structural and computational studies of the potassium channel T1 domain and the ligand binding domain of a glutamate receptor /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3036994.

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34

Lu, Zhisheng. "Structural studies of HIV-1 Vif and its SOCS-box domain". Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/structural-studies-of-hiv1-vif-and-its-socsbox-domain(710df582-c838-4935-b6f6-c023195997c7).html.

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The human immunodeficiency virus type I (HIV-1) is a retrovirus that damages the human immune system, which is suppressed by a cellular factor APOBEC3G in non-permissive cells. The viral infectivity factor (Vif) can induce a poly-ubiquitination degradation of APOBEC3G to counteract the immune response by forming an E3 ubiquitin complex composed of cellular proteins Elongin B (EloB), Elongin C (EloC), Cullin 5 and Ring-Box protein. In this project, we solved the first structure for the Vif SOCS-box and EloBC complex in solution by Nuclear Magnetic Resonance, which shows that the proline-rich motif in the SOCS-box binds to the EloB carboxyl terminus by backbone interaction, based on weak van der Waals forces. Based on the results from cell assays it shows that the residues on the Vif proline-rich motif play an important role in viral infectivity; the proline-rich motif induced structure of the C-terminal tail has been demonstrated to be critical for EloB to perform further biological function, by a mechanism which allows HIV-1 to evade the immune response. In addition, expression and purification on full-length Vif in the presence of EloBC and core binding factor β (CBFβ) has been performed. These studies showed that soluble Vif in the tetramer is achieved when it is expressed at a low temperature with a low IPTG concentration. Solubility tests indicate that this complex must be kept in high salt concentration in order to prevent self-association. Comparison with previous studies on Vif expression and purification. This protocol enables one to obtain purified Vif from E. coli and opens the way to solve the structure by X-ray crystallography and extend our understanding on the Vif-induced APOBEC3G degradation mechanism.

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35

Plans, Calafell Vanessa. "Studies on RNF8, a ubiquitin ligase with a RING finger domain". Doctoral thesis, Universitat de Barcelona, 2005. http://hdl.handle.net/10803/1868.

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La ubiquitinació és una modificació postraduccional de proteïnes empleada en la regulació de molts tipus diferents de senyalitzacions cel·lulars i processos biològics. Els enzims responsables d'aquesta modificació postraduccional són les E1 o enzims d'activació de ubiquitines, les E2 o enzims de conjugació de ubiquitines i les E3 o lligases de ubiquitines i poden conjugar una o més ubiquitines sobre la proteïna substrat.
L'heterodimer format per UBC13-UEV te activitat de conjugació de ubiquitines mitjançant enllaços que utilitzen la lisina en posició 63 de la ubiquitina en contes de la lisina en posició 48. Aquesta modificació no es reconeguda pel proteasoma i per tant no implica una degradació de la proteïna que la porta. La cerca de proteïnes que interactuen amb UBC13 ens va permetre de identificar dues proteines: RNF8 i KIAA0675 que contenen un domini RING finger, pel qual interactuen amb UBC13. RNF8 te activitat autocatalítica de lligasa de ubiquitines, el que li permet elongar cadenes de poliubiquitines tant per lisina 48 com per lisina 63; aquesta darrera depèn de l'activitat catalítica de UBC13. A més, RNF8 co-localitza amb UBC13 en estructures nuclears.
L'estructura en dominis d'RNF8 és la mateixa que la del regulador del checkpoint mitòtic CHFR, ambdues proteïnes tenen un domini RING finger de reclutament d'enzims de conjugació de ubiquitines, al extrem amino terminal i un domini FHA de reconeixement de fosfopèptids. RNF8 localitza en el nucli cel·lular i te un patró d'expressió depenent de cicle amb uns nivells proteics que augmenten progressivament de G1 a M, assolint un màxim en metafase seguit d'una brusca desaparició en anafase i una recuperació de l'expressió de la proteïna en fases més tardanes de la mitosi. La sobre-expressió de RNF8 causa una reducció de la població en G2-M i una acumulació de cèl·lules en G1. La depleció d'RNF8 mitjançant la tècnica de RNAi no causa cap efecte significatiu sobre el cicle cel·lular basal. En canvi, la depleció d'RNF8 causa un retard en la sortida de mitosi després d'un arrest induït per nocodazole, fet que s'associa amb una reducció del turnover de la ciclina B1, que és un substrat de l'APC/C. Recíprocament, la sobre-expressió de RNF8 impedeix a les cèl·lules d'acumular-se en G2-M en resposta a un tractament amb nocodazole. A més l'activació del Spindle Assembly Checkpoint (SAC), mitjançant un breu tractament amb nocodazole, en combinació amb la sobre-expressió d'RNF8 impedeix que la proteïna del checkpoint Mad2 es localitzi als quinetocors. Aquesta funció d'RNF8 depèn de l'activitat lligasa ja que un mutant d'RNF8 incapaç de conjugar ubiquitines produeix uns efectes significativament atenuats en comparació amb la proteïna salvatge. Aquestes observacions suggereixen que RNF8 és un regulador negatiu del SAC i permet la reactivació de l'APC/C en condicions d'estrès mitòtic.
A més, RNF8 presenta activitat pro-apoptòtica. L'expressió ectòpica d'RNF8 en cèl·lules HeLa activa les caspases-3 i -8 i l'activitat pro-apoptòtica resultant es pot inhibir mitjançant la incubació amb els inhibidors de caspases ZVAD i ZDEVD. La mort cel·lular induïda per RNF8 es veu atenuada en gran mesura per la sobre-expressió del mutant d'RNF8 que no te activitat lligasa de ubiquitines. El tractament amb diferents agents genotòxics provoca una acumulació dosi-depenent de la proteïna RNF8 endògena que no es correlaciona amb un increment dels nivells de transcrit. Finalment, la depleció d'RNF8 permet a les cèl·lules HeLa de ser més resistents al tractament amb etopòsid, suggerint que RNF8 te un paper important regulant la mort cel·lular causada per aquesta droga.
Ubiquitylation is a post-translational protein modification, which regulates very different signaling pathways and biological processes. Ubiquitylation occurs thanks to the catalytic activity of ubiquitin activating enzymes or E1, ubiquitin conjugating enzymes or E2 and ubiquitin ligases or E3 and the substrate can be modified with one or more ubiquitin moieties that form polyubiquitin chains.
The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified two proteins, namely RNF8, KIA00675, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2's that mediate canonical (K48) polyubiquitylation. RNF8 has ubiquitin ligase autocatalytic activity that elongates chains through either K48 or K63 of ubiquitin, and UBC13 activity is required to elongate the latter chains. RNF8 and UBC13 co-localize in the nucleus.
RNF8 is a ubiquitin ligase that bears a FHA domain near its amino end, and a RING finger domain at its carboxy terminus, through which it can recruit several ubiquitin-conjugating enzymes. In metazoans, only the mitotic checkpoint regulator CHFR shares this domain architecture. RNF8 follows a cell-cycle-dependent turnover, with levels increasing from G1 to M, reaching a maximum at metaphase, followed by an abrupt decline at anaphase, and recovery of protein levels at the end of mitosis. Overexpression of RNF8 caused a reduction of the G2-M population and an accumulation of cells in G1, while siRNA depletion did not significantly alter the basal cell cycle. Depletion of RNF8 caused a delay in the exit from nocodazole-induced arrest, which was associated with a reduced turnover of the APC/C substrate cyclin B1. Conversely, cells overexpressing RNF8 failed to arrest at G2-M upon treatment with nocodazole. In addition, activation of the spindle assembly checkpoint with a brief exposure to nocodazole in combination with RNF8 overexpression prevented the localization of Mad2 to kinetochores. These functions of RNF8 were dependent on its ubiquitin ligase activity, since a ligase-inactive mutant produced significantly attenuated effects as compared to the wild-type protein. Taken together, these observations suggest that RNF8 is a negative regulator of the spindle assembly checkpoint that reactivates the APC/C under conditions of mitotic stress.
In addition,RNF8 functions as a proapoptotic protein. Ectopic expression of RNF8 in HeLa cells induced the activation of caspases 3 and 8, and the ensuing apoptosis was prevented by incubation with the caspase inhibitors ZVAD and ZDEVD. The apoptotic death induced by RNF8 was greatly attenuated by introduction of a point mutation in its RING finger domain that rendered it non-functional as a ubiquitin ligase, indicating that this function is essential for the proapoptotic activity of RNF8. Treatment with DNA-damaging agents causes a dose-dependent accumulation of endogenous RNF8 without increasing its mRNA levels. Finally, depletion of RNF8 with specific siRNA duplexes effectively prevented the apoptosis and caspase-3 up regulation induced by the topoisomerase II inhibitor etoposide, suggesting that RNF8 plays a major role in regulating apoptotic death caused by DNA damaging agent.

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36

Salek, Reza M. "NMR studies of the heat shock protein 90 N-terminal domain". Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446807/.

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The Hsp90 based chaperone is a ubiquitous protein-folding system in the cytoplasm of eukaryotes. Several signal transduction systems and cell cycling pathways utilise an interaction with Hsp90 as an essential component. The Hsp90 chaperone is an ATP dependent chaperone, which is active as a dimer. The N- terminal domain of Hsp90 itself has very weak ATPase activity and plays an essential role in the mechanism of dimerisation. This study attempts to elucidate the nucleotide binding effects on the Hsp90 N-terminal domain by NMR. Accomplishing backbone assignments of the apo- and AMP-PNP bound forms provided a system for which individual residues could be investigated. About 200 backbone amide peaks in the HSQC were observed out of a reported 207 residues for the Hsp90 N-terminal domain. Out of these, 192 for the apo- and 182 for the AMP-PNP bound forms were assigned. Assignments were also obtained for the HN, N, C, and CO nuclei. Comparison of apo and AMP-PNP HSQC spectra showed large shift differences in areas where the nucleotide binds and in a conserved loop, which has been proposed to act as a lid to the active site. The chemical shift pattern of the AMP-PNP bound form compared to that of the ADP bound form showed a different local environment for at least 30 residues, suggesting that different nucleotide bound conformations are more than just nucleotide structural differences. Analysing the NMR results suggests that binding of AMP-PNP to the Hsp90 N-terminal domain is not sufficient to cause lid closure as previously thought. Relaxation studies highlighted regions that had different local motions in the apo- and nucleotide bound form based on individual residues within the Hsp90 N- terminal domain. The protein rotational correlation time was measured at 12.5 ns in the apo and 14 ns in the AMP-PNP bound form. No interaction between the labelled N-terminal domain and non-labelled middle domain of Hsp90 was observed. The antibiotic, novobiocin, which inhibits other members of the GHKL superfamily, was also shown not to bind to the N-terminal domain, consistent with previous studies. The study of two mutants, A107N and T101I, showed the effects of mutation in the lid region of the N-terminal domain causing chemical shifts of between 20-30 residues. Neither of these two mutants were able to bind AMP-PNP. In all nucleotide bound X-ray crystal structures solved to date, no difference between the ADP and ATP bound forms has been observed and only one conformation was found. The results presented here suggest that the structure in solution is much more variable than previously envisaged.

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37

Marigheto, Niusa A. "Time-Domain NMR Studies of the Internal Quality of Horticultural Products". Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485289.

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Nuclear Magnetic Resonance was used to investigate selected fruit with internal defects and at different stages of ripening. This was motivated by the need for an industrial NMR sensor of fruit and vegetable quality capable of operating at typical industrial conveyor speeds. Such a sensor requires the development of NMR protocols capable of determining fruit and vegetable quality in a single-shot manner. It also requires knowledge of which combination of NMR parameters is most sensitive to the quality factor of interest. In this thesis we therefore address this issue by exploring the off-line relationship between NMR parameters and fruit quality. We mainly focus on three areas: the detection of mealiness in apples, the single-shot measurement of Brix, and a single-shot measurement of oil in avocado. One dimensional techniques, measuring transverse relaxation times, were used as starting point. The correlation between mealiness and one dimensional was not observed at low fields. Therefore two-dimensional approaches were taken. The first of these was correlation spectroscopy, which showed that the of the water associated with the cell wall in mealy apples is much longer that that of fresh apples. This in principle could be used to classify apples. The differences between fresh and mealy apples observed with the second two-dimensional protocol, could ~ot be used to classify apples. However, when the technique was applied to avocado, it became clear that the difference between the diffusion coefficients of water and oil could be exploited for an on-line application. A fast pulse sequence was developed and the off-line measurements showed a good correlation between oil content and the echo ratio. A similar protocol was developed for the on-line measurement of Brix value in apple and strawberry.

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38

Hart, Christopher T. "Functional and Structural Studies of the Anti- MinCD Domain of MinE". Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28643.

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The Min protein system, comprised of MinC, MinD and MinE, functions to ensure that the cytokinetic septum formed during bacterial binary fission is placed at midcell in gram negative bacteria. When bound to ATP, MinD binds to the cell membrane and recruits MinC, forming a complex which inhibits formation of the cell division septum. MinE plays a central role in the regulation of this process by opposing this inhibition through interactions with MinD that displace MinC and site-selectively permit formation of the cell division septum at midcell. However, the amino acid residues of MinE that are important for this interaction have yet to be delineated. In this thesis, I present data from an in vitro ATPase assay where I examined the ability of MinE mutant proteins from Neisseria gonorrhoeae ( N. gonorrhoeae), and synthetic peptides corresponding to the N-terminal amino acids of N. gonorrhoeae, to stimulate the ATPase activity of MinD from N. gonorrhoeae. The in vitro experimental data suggests that a sequence of five amino acids in the N-terminus of MinE plays a crucial role in MinE's ability to stimulate MinD ATPase activity and suggests that the N-terminal region of MinE is a functionally autonomous domain. To support the in vitro data, I also present preliminary data from an in vivo assay examining select MinE mutants. The preliminary in vivo experimental data supports the in vitro data, suggesting the importance of five amino acids in the N-terminus of MinE. Finally, I present spectroscopic data to complement my findings from both in vivo and in vitro assays. The results from this study have implications for all current theories regarding the mechanism by which MinE ensures that cell division occurs exclusively at midcell in gram negative bacteria.

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39

Cheng, Shan Amy. "Structure-function studies of secreted PDZ domain-containing protein 2 (sPDZD2)". Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558101.

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40

Dutnall, Robert Nicholas. "Structural and functional studies of a zinc finger DNA-binding domain". Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360030.

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41

Wilson, Gillian Mary. "High frequency dielectric studies of cell suspensions using time domain reflectometry". Thesis, Bangor University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305946.

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42

Fowler, S. B. "Folding studies on an immunoglobulin domain using chemical and mechanical methods". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599147.

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Part I of this thesis focuses on the folding and stability of TI 127, the 27^th Ig domain from the I-band of human cardiac titin, using chemical denaturants. The first comprehensive protein engineering analysis of the transition of an immunoglobulin (Ig) I-set domain is presented. Preliminary studies reveal that TI 127 folds via a kinetic intermediate. The transition state shifts to a less native-like state upon mutation, an indication of strain in the hydrophobic core. These two effects make a standard analysis impossible. However, the data can be used to identify critical residues involved in the transition state for folding. The transition state structure is compared with those of three other Ig-like domains from different evolutionary superfamilies and with little sequence homology. Results show that for all four domains, folding is driven by a ring of interacting residues in the common structural core. This indicates that the folding pathway is driven by thermodynamic features of the fold rather than evolutionary constraints. The I-band of titin is composed principally of Ig domains arranged in tandem and is subjected to applied force in fulfilling its function in vivo. These domains must resist force and also be able to unfold and recover. Atomic force microscopy (AFM) is a relatively new tool for studying protein unfolding in the presence of applied force. In part II a detailed analysis of the unfolding of TI 127 is presented and compared with results form Part I. Under applied force, TI 127 unfolds via a stable intermediate and a model for its structure is proposed. Protein engineering analysis shows that the loss of the same set of interactions in the structure initiates unfolding using both denaturants and AFM. If unfolding under applied force reflects events in vivo, then this indicates that kinetic information from studies using denaturants may be directly applicable to in vivo behaviour.

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43

Ford, M. G. J. "Structural and functional studies on ANTH and EUTH domain-containing proteins". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599114.

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In this work, the crystal structure of the NH₂-terminal domain of CALM, the ubiquitous isoform of AP180, is presented, both alone and bound to various inositol polyphosphates and phosphatidylinositol-4, 5-bisphosphate (PtdIns(4,5)P₂). The domain has a novel, all-helical fold which we name the AP180like NH₂-terminal homology (ANTH) domain. The ligands bind to the surface of the structure, to a lysine-rich motif. This lysine-rich motif was confirmed as the binding site by biochemical means and binding to PtdIns(4,5)P₂ is responsible for recruiting full-length AP180 to membranes. The COOH-terminal domain of AP180 is known to bind clathrin and to stimulate clathrin lattice assembly. By simultaneously binding membranes containing PtdIns(4,5)P₂ and clathrin, it is shown that AP180 can recruit clathrin to membranes. Using a novel assay where a lipid monolayer is used as a membrane mimetic, it is shown that AP180 can stimulate the polymerisation of the recruited clathrin. The resulting clathrin lattices are predominantly flat and are homogeneous in size. In the additional presence of AP2, invaginated lattices are formed. Database screens for proteins containing the lysine-rich motif identified Huntingtin Interacting Protein 1 Related (HIP1R). The NH₂-terminal domain of HIPIR shares some (~20%) sequence identity with the AP180 ANTH domain. Indeed, the crystal structure of the NH₂-terminal domain of HIPIR has a fold which is very similar to that of the CALM ANTH domain, showing that it, too, is an ANTH domain. HIPIR ANTH interacts with PtdIns(4,5)P₂-containing membranes via its lysine rich motif. Together these observations suggest that ANTH domains may be responsible for recruiting host proteins to membranes containing PtdIns(4,5)P₂. The epsin NH₂-terminal homology (ENTH) domain of epsin is structurally similar to the ANTH domain - the first 7 helices superpose well - though it lacks the lysine-rich motif. However, the ENTH domain also binds to inositol polyphosphates and to membranes containing PtdIns(4,5)P₂.

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44

鄭珊 i Shan Amy Cheng. "Structure-function studies of secreted PDZ domain-containing protein 2(sPDZD2)". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558101.

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45

Jacks, Amanda Jane. "NMR studies of the starch-binding domain of Aspergillus niger glucoamylase". Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364191.

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46

McIntosh, Pauline Bernadette. "NMR based studies of the DNA-binding domain from B-Myb". Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388299.

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47

Klemm, Juli Dawn. "Structural and biochemical studies of Oct-1 POU domain-DNA interactions". Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/32667.

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48

Shu, Ningcheng. "Structural studies of biotinyl domain of E. coli acetyl-CoA carboxylase". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620559.

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49

McIntosh, Lisa. "Development and characterisation studies of a type III fibronectin domain pair". Thesis, University of Strathclyde, 2014. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=24607.

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This thesis presents an investigation into a 9th-10th type III fibronectin (FN) domain pair (FIII9-10) focused on the characterisation and functional activity of the protein. Initial work attempted to establish whether there was a binding interaction between FIII9-10 and IGFBP-5. Surface plasmon resonance failed to demonstrate an interaction and so thereafter the programme examined the use of FIII9-10 as a protein scaffold for cell adhesion. Surface-induced unfolding of proteins results in a loss of function. To improve the conformational stability of FIII9-10, Pro-Pro pairs were introduced. A previously developed multimeric FIII9'10 α5β1 ligand was also studied. FIII9-10 cDNA constructs were expressed in E. coli. Purity, fold and molecular weight were confirmed using SDS-PAGE, circular dichroism (CD) and mass spectrometry. Changes in conformational stability generated by the mutations were assessed by equilibrium chemical denaturation. An automated CD system was used to follow the secondary structure changes generated by thermal denaturation. Adsorption induced structural changes were investigated using a novel 'solid state' CD technique for a range of surface energies. Neutron reflectometry was employed to estimate the surface coverage and packing arrangement of the FIII9-10 mutants. Calculation of the Gibbs free energy demonstrated a two-fold increase in stability compared to wild type with a 9-11°C increase in the Tm. The mutated proteins showed enhanced stability when adsorbed to silica, but lost structure as surface hydrophobicity increased. This loss was less than that for the wild-type. ELISA studies verified that the mutation of the FIII9-10 did not compromise the receptor binding affinity. Immunofluorescence microscopy of baby hamster kidney on protein coated substrates displayed improved cell adhesion and spreading. The enhanced structural stability of the FIII9-10 mutants showed promise for use in influencing and controlling the interactions between medical implants and host tissue, mimicking the role of the extracellular matrix and improving biocompatibility.

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50

Hanson, Sonya M. "Structural, biochemical and computational studies of TRP channel transmembrane domain modularity". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:328269a9-11c0-4d5b-9cb7-d7433cf4d6c4.

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Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [³H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.

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