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Whitehead, L. "Computer simulation of biological membranes and membrane bound proteins." Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297412.
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LADHA, PARAG. "POLYMERIC MEMBRANE SUPPORTED BILAYER LIPID MEMBRANES RECONSTITUTED WITH BIOLOGICAL TRANSPORT PROTEINS." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1145901880.
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Boulter, Jonathan Michael. "Structural and functional studies of the erythrocyte anion exchanger, band 3." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297079.
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Dewolf, Christine Elizabeth. "Properties of model biological membranes." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244082.
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Parton, Daniel L. "Pushing the boundaries : molecular dynamics simulations of complex biological membranes." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:7ab91b51-a5ae-46b4-b6dc-3f0dd3f0b477.
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A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.
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Enders, Oliver. "Structural analysis of biological membranes and proteins by atomic force microscopy." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972570497.
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Moës, Elien. "Theiler's murine encephalomyelitis protein 2C and its effect on membrane trafficking." Thesis, St Andrews, 2008. http://hdl.handle.net/10023/540.
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Rhonemus, Troy A. "Reagents for protein analysis and modification." Virtual Press, 1998. http://liblink.bsu.edu/uhtbin/catkey/1115753.
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Spelbrink, Robert G. "The role of the yeast GRD20 protein in membrane trafficking and actin organization /." free to MU campus, to others for purchase, 2000. http://wwwlib.umi.com/cr/mo/fullcit?p9974686.
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Millman, Jonathan Scott Andrews David. "Characterization of membrane-binding by FtsY, the prokaryote SRP receptor /." *McMaster only, 2002.
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Yin, Zhao. "Characterization of the biological function of AtEXO70E2." HKBU Institutional Repository, 2018. https://repository.hkbu.edu.hk/etd_oa/483.
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Exocyst positive organelle (EXPO) is a newly discovered double membrane organelle involved in exocytosis and likely other vesicle trafficking processes. EXPO is likely generated from the ER, fused with plasma membrane and released a single membrane vesicle to cell exterior. The Arabidopsis protein Exo70E2 was found to be associated with EXPO and therefore is considered as a marker of EXPO and might play a role in EXPO-mediated vesicle trafficking. Understanding the biological function of AtExo70E2 (abbreviated as E2 in this thesis) will be very helpful in unraveling the function of EXPO. The aim of this work was to use various molecular, genetic and physiological approaches to determine the possible role of Arabidopsis Exo70E2 in biological pathways. By using the Exo70E2pro:GUS line, the expression pattern of Exo70E2 was determined. Exo70E2 was expressed mainly in roots, especially in root tips and epidermal cells in the division and elongation zones of roots. Its expression level was induced when the seedlings were treated with Flg22, a peptide derived from bacterial flagillin protein that induces the plant defense response. The tissue subcellular localization of Exo70E2 was also studied using the 35S:Exo70E2-eYFP and Exo70E2pro:Exo70E2-GFP reporter lines. The GFP fusion protein was found primarily in the epidermal cells of roots even in the 35S:Exo70E2-eYFP lines. For phenotypic analysis resulting from mutations of the Exo70E2 gene, I obtained three T-DNA insertion mutant lines and generated its overexpression lines. The two mutant alleles, e2-2 and e2-3 are in the Columbia ecotype background and further characterized. e2-2 which has a T-DNA insertion in an exon is likely a knock out line as Exo70E2 gene transcript could not be detected. e2-3, which carries a T-DNA insertion in its promoter region, was found to accumulate a higher level of the transcript, suggesting that the insertion causes its enhanced expression of Exo70E2. There was no obvious difference between wild type and e2-2 in their phenotypes under different conditions tested in this study. However, e2-3 had a retarded growth phenotype when grown in soil or on MS medium. The seedlings of e2-3 on MS medium also had a yellowish color although such a phenotype was not obvious when they were grown in soil. When supplementing the MS medium with sucrose, glucose or mannitol, the growth of e2-3 was more reduced compared to wild type under these conditions. However, on the medium with NaCl or under phosphate deficiency, the yellowish phenotype of e2-3 was rescued and the mutant seedlings became relatively healthier than the seedlings under the regular MS medium. A proteomics approach was taken to compare protein secreted from the seedlings of wild type and the mutants. Proteins secreted by seedlings to the liquid medium were collected, concentrated and subjected to MS analysis. Comparison of the profiles of secreted proteins between the wild type and the mutants leaded to identification of candidate proteins whose secretion might be affected by the mutation. My study indicates that Exo70E2 and EXPO are involved in transporting proteins (likely also metabolites) to the exterior of cells and the rhizosphere and might play an important role in stress responses.
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Liu, Yu. "Biological properties of EBV-encoded latent membrane protein 1 in nasopharyngeal epithelial cells /." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23001008.
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Abuillan, Wasim [Verfasser], and Motomu [Akademischer Betreuer] Tanaka. "Fine-Structures, Lateral Correlation and Diffusion of Membrane-Associated Proteins on Biological Membrane Surfaces / Wasim Abuillan ; Betreuer: Motomu Tanaka." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://d-nb.info/1177809621/34.
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Botero-Kleiven, Silvia. "Identification of new proteins and biological processes in the apicomplexan Toxoplasma gondii /c Silvia Botero-Kleiven." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-983-1/.
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Quiroga, Álvarez Xarxa. "Plasma membrane mechanosensing upon stretch-induced topography remodelling." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/672367.
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Five years ago, I started walking this path that now seems like an entire life. Although everyone around tried to explain how this would feel, none of their explanations could have match what it has been in the end. And this is exactly how living systems are, at all levels. The harder the scientists try to feed our curiosity taking closer looks to them, inspecting the question from a different angle, and despite all the previous knowledge that we could gather; the more they surprise us and reveal new ways of sensing, reacting and adapting to the environment. And I think this is exactly what drove me here. I wanted to understand how this is done. I wanted to “see” it. How is it possible that a cell “understands” what is going on around? Which are the parameters that they sense? Biochemistry alone does not answer the question.
In a crowded environment, such as it is our body, cells are exposed to thousands of mechanical stimuli too. And those can be also harnessed and transformed into chemical responses as a way of signalling. While the classical biochemical inputs have long been studied, loads of questions remain open about how cells interpret those physical stimuli around them, and microscopy comes as a powerful technique to try to answer these queries.
In that sense, this thesis represents a small approach in trying to unravel how the plasma membrane, the first boundary between the cell and the extracellular media, can receive mechanical stimuli and convert them into biochemical signals amenable for the cell. To try to answer this question, this work starts with an introduction to the structure and physicochemical characteristics of the plasma membrane. An overview of the cortical component of the cytoskeleton, intrinsically interconnected to this structure, is also provided. Next, a summary of the literature available on how the plasma membrane can perceive mechanical
stimuli and which are the associated biochemical responses triggered by them is included as well. This part is based on a review article published by my colleague and co-supervisor Dr. Le Roux and myself at Philosophical Transactions B as part of the 2019 issue “Forces in cancer” [1].
After the introduction, chapter 2 describes the objectives of this study, which can be summarised as trying to unravel the way in which cells couple mechanical signals at their plasma membrane to biochemical cascades that mediate a response to those. Following, chapters 3 and 4 compose the main body of this thesis, including the methods and the experimental results coming from this research work. Both sections constitute a scientific article that has been recently submitted for publication. In chapter 3 the simplified model chosen to study the question of how cells sense and integrate mechanical stimuli at their plasma membrane is described. This consisted in submitting fibroblast to a controlled stretch-release cycle, forcing them to quickly adapt their shape, mimmicking a highly-relevant scenario in physiology. Chapter 4 recapitulates the way in which plasma membrane reacted to this mechanical perturbation. In the first place, the structure reacted by passively forming evaginations on the nanometric scale of homogeneous size and shape. These evaginations are next recognised by the I- BAR protein IRSp53, which subsequently organizes a node of actin polymerisation dependent on Rac1 and Arp2/3 that mediates the flattening of the structures. Absence of IRSp53 results, thus, in an impaired recovery of homeostasis after stretch. To reinforce the obtained experimental results, theoretical framework to model the mechanics of the system was generated in collaboration with the group of Dr. Arroyo at the Centre Internacional de Mètodes Numèrics en Enginyeria (CIMNE). The aim of this model was to describe how a network generated by the Arp2/3 complex, until now described to push, is able to generate in-plane forces that mediate the ironing of the evagination. Chapter 5 includes a discussion about the limitations of the technique, the novelty of the presented findings, the possible physiological scenarios where the described mechanochemical pathway can be of relevance and, finally, some exciting and unexplored questions that remained open after this work.
Additionally, other scientific production obtained during this thesis consisting in unexplored results or work belonging to other publications, has been added at the end of this manuscript in four appendixes. On the first one, I describe all the efforts made during the first 1 year and a half of the PhD to improve immunostaining technique for plasma membrane bound proteins in order to try
to stain endogenous BAR proteins. The second appendix gathers the findings obtained from the silencing assay of BAR candidates likely to recognise the curved shape of the stretch-release generated evaginations. The third appendix contains experimental results part of a different publication from Le Roux et al. now under review in Nature Communications [2]. Here, I studied the response of the N-BAR protein Amphiphysin after mechanical stimulation in cells. A fourth appendix including scanning electron microscopy representative images of stretch-release generated evaginations in other cell lines is also added. Finally, I included two more appendix containing the sequencing of all the IRSp53 plasmids used for the body of the work of this thesis and the MATLAB code used for analysis of evaginations flattening dynamics after stretch.
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GLADDING, SARAH M. "POROUS INORGANIC SUPPORTED LIQUID MEMBRANES FOR USE IN ION CHANNELING." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1109343185.
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Jaburek, Martin. "Kinetics and regulation of mitochondrial cation transport systems /." Full text open access at:, 1999. http://content.ohsu.edu/u?/etd,207.
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Jiang, Fenglei. "Structure/function mapping studies of the E. coli YIDC." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1055436619.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xv, 119 p. ; also includes graphics (some col.). Includes bibliographical references (p.111-119). Available online via OhioLINK's ETD Center
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Liu, Yu, and 劉鈺. "Biological properties of EBV-encoded latent membrane protein 1 in nasopharyngeal epithelial cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31242078.
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Ugarte, La Torre Diego Renato. "Force field development for performing coarse-grained molecular dynamics simulations of biological membranes." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265177.
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Zhu, Lin. "Structural studies of HDL and applications of EM on membrane proteins." Doctoral thesis, KTH, Strukturell bioteknik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-204045.
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A large number of proteins interact with biological membranes, either integrated in the membrane (PepTSo2), embedded on a membrane surface (5-lipoxygenase) or encircling a cutout of lipid bilayer (apolipoprotein1 (apoA-I). They function as transporters, receptors or biocatalysts in cellular processes like inflammation or cholesterol transport which are touched upon here. Malfunction of specific membrane proteins are the cause for several diseases or disorders. Knowledge of protein structure supports understanding of its mechanism of function. Here, transmission electron microscopy (TEM) was used for structure determination. To obtain structure information to high resolution for membrane proteins, normally surrounded by lipids, demands specific methods and materials for stabilization. Stabilized in detergent the structure of the bacterial transporter PepTSo2 was shown to form a tetramer even bound to substrate. However, with a protein based stabilizer, Salipro, the structure of PepTSo2 could be determined to high resolution. High density lipoprotein (HDL) in blood plasma, involved in the removal of cholesterol from peripheral tissues, have a central role in cardiovascular function, metabolic syndrome and diabetes. The HDL-particle is composed of two copies of ApoA1 and around hundred lipid molecules. From TEM data, for the first time the clearly discoidal shape could be shown by 3-dimendional reconstructions. These were used for modelling the ApoA1 protein dimer by a "biased fitting" procedure. The results indicate how ApoA1 folds around a lipid bilayer in a disc-shaped structure. Modified HDL called nanodiscs were here used to show the Ca2+ dependent binding of 5-lipoxygenase on the nanodisc bilayer and thereby increased production of the inflammatory mediator leukotrieneA4. Dimerization of 5-lipoxygenase inactivates these functions.QC 20170323
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Bruinsma, Paul. "The role of the yeast COG3, VPS35, and YDR141C proteins in membrane trafficking /." free to MU campus, to others for purchase, 2002. http://wwwlib.umi.com/cr/mo/fullcit?p3074381.
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Williams, Taufika Islam. "METHODS DEVELOPMENT IN BIOLOGICAL MASS SPECTROMETRY: APPLICATIONS IN SMALL MOLECULE RESEARCH AND PROTEOMICS." UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/288.
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Technological developments have enabled mass spectrometry (MS) to evolve asone of the most versatile, sensitive and widely used analytical methods. Key areas ofresearch in biological MS include the development of analyte-selective MSmethodologies, along with the design of MS compatible separation technology. Analytesof interest range from small, biologically active molecules in disease progressionresearch, to macromolecules such as proteins, in proteomics investigations. Advances inthese areas are vital to maintaining the level of sophistication that has become thebenchmark for MS analyses.Mass spectrometry has found a permanent station in disease progression studies,particularly in biomarker discovery. This is especially true for Alzheimer's disease (AD),a condition marked by widespread lipid peroxidation (LPO) in the brain. The mainhypothesis of the first part of this dissertation is that LPO produces aldehydes that canpotentially be exploited as AD biomarkers. Design of novel LC-MS/MS methods forbrain aldehyde analysis is described. The methods were applied towards aldehydequantification in the hippocampus, superior and middle temporal gyrus and cerebellum ofsubjects with early AD (EAD), mild cognitive impairment (MCI) and age-matchedcontrols. Results obtained indicated elevation of neurotoxic aldehydes in MCI and EADbrain and suggested that LPO occurred early in AD. Understanding AD progression hasbecome important for developing diagnostic methods and treatments.Mass spectrometry is also the major analytical tool in proteomics, where gelelectrophoresis is dominant in pre-MS separations. The main hypothesis of the latter partof this dissertation is that exposure of microbe fermenters including Clostridiumthermocellum to an external stimulus, such as ethanol, can alter the membrane proteome.Design of novel doubled-SDS-PAGE (dSDS-PAGE) methods for membrane proteinanalysis is described, as these proteins are under-represented in standard 2D-PAGE. Thenewly developed Bicine-dSDS-PAGE offered superior separation over other methods andwas applied towards analysis of wild type and ethanol-adapted C. thermocellum cellmembranes. Significant differences in protein expression were observed. Anunderstanding of ethanol adaptation will promote the design of more ethanol-tolerantstrains. Such an outcome can have dramatic effects in the fuel industry as the trendtowards more efficient fuel development gathers momentum.
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Brault, Jeffrey J. "Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types." free to MU Campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p3091902.
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Graham, John Stephen. "Mechanical properties of complex biological systems using AFM-based force spectroscopy." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/4191.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.
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Bain, Evelyn Louisa. "The physics of biological systems : Part (i), The circadian systems of neurospora crassa; Part (ii), The diffusion of proteins in biological membranes." Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404831.
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Stanley, Nathaniel H. 1983. "Understanding disordered and membrane protein recognition by molecular dynamics." Doctoral thesis, Universitat Pompeu Fabra, 2015. http://hdl.handle.net/10803/384535.
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This thesis has been about the use of a simulation technique, known as molecular dynamics simulations, to study biophysics in proteins that have historically been difficult to study with other methods. We have studied numerous systems, namely binding to the membrane proteins Fatty acid amide hydrolase (FAAH) and the sphingosine-1-phosphate receptor 1 (S1P1R), and folding in the disordered protein kinase inducible domain (KID). In each case we have been able to analyze processes and uncover behaviors that are difficult or impossible to view by other means.Esta tesis se trata del uso de un técnica de simulación, llamado simulación de dinámica molecular, para estudiar la biofísica de proteínas que históricamente han estado difícil estudiar por otros métodos. Hemos estudiado numerosas sistemas, en particular la encuadernación de ligandos en sistemas de membranas como Fatty acid amide hydrolase (FAAH) y el receptor Sphingosine-1-phosphate (S1P1R), y cómo se pliegue una proteína desordenada llamado Kinase inducible domain (KID). En cada caso hemos sido capaz de analizar procesos y detapar comportamientos que sean difícil o imposible ver por otros métodos.
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Lo, Kwok-fung Angela, and 勞幗鳳. "Alterations of gene expression and biological properties in nasopharyngeal epithelial cells by the Epstein-barr virus encodedlatent membrane protein 1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31243423.
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Krylov, Dmitri M. "Guanylyl cyclase activating protein-1 and its regulation of retinal guanylyl cyclases : a study by molecular biological methods and a novel mass spectrometry based method /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9259.
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Suárez, Germà Carme. "Investigation of the phospholipid peripheral region of lactose permease in model membranes." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/125470.
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The interaction between a membrane protein and its surrounding phospholipids is thought to be crucial for the correct folding and function of the protein. This thesis is focused on the investigation of the interplay between Lactose permease (LacY), a paradigm for secondary transporters present in the inner membrane of Escherichia coli and model systems mimicking its natural lipid environment. Since the role of phospholipids in LacY’s activity is currently being refined, this work represents a contribution to the field by studying the interaction at two different levels: (i) the LacY interplay with the phospholipids present at the annular region in the vicinity of the protein was studied through FRET measurements between a single-tryptophan LacY mutant and diverse pyrene-marked phospholipids, and (ii) the LacY interaction with the more distanced bulk phospholipids was studied through supported proteo-lipid sheets that were analysed using topography, force-spectroscopy and force-volume Atomic Force Microscopy modes. First, after validating LacY preference for phospholipid fluid (Lα) phases in the studied two-component model systems, a different composition between bulk and annular regions was confirmed. Hence, bulk lipids, which were assimilated to the phospholipids in Lα phase, were mainly formed by PG, while PE was the main component of the annular region. This points to a direct annular phospholipid-LacY selectivity because it discards a random phospholipid distribution near the protein. Second, the LacY selectivity for precise phospholipid species at the annular region was found to be related to: (i) a neutral charged phosholipid (PE or PC, with preference for the former), and (ii) phospholipids with large negative spontaneous curvature (C0) (DOPE > POPE). In addition, D68 was revealed as an important amino acid for the protein annular lipid selectivity. Third, the interaction between LacY and the bulk lipids was described as reciprocal. Accordingly, the presence of the protein largely modified the topography and the nanomechanics of the lipid system, especially for the Lα phase, whilst the nanomechanics of LacY itself were different depending on the surrounding lipid matrix: more force was needed to pull LacY form the DPPE:POPG (3:1, mol/mol) system than from the POPE:POPG (3:1, mol/mol) one. Therefore, the bilayer lipid composition seems to determine the forces governing the LacY tight interaction with the membrane and can be thus decisive for the protein correct insertion and activity.La interacció entre una proteïna de membrana i els fosfolípids que l’envolten és crucial pel bon plegament i la correcta funció de la proteïna. Aquesta tesi està centrada en la investigació de la interacció entre la Lactosa permeasa (LacY), un paradigma dels transportadors secundaris situat a la membrana interna d’Escherichia coli, i sistemes models que mimetitzen el seu entorn lipídic. Aquest treball representa una contribució al camp a través de l’estudi de la interacció a dos nivells: (i) la interacció entre LacY i els fosfolípids presents a la regió anular propera a la proteïna ha estat estudiada a través de mesures de FRET entre un mutant de LacY amb un únic triptòfan i diversos fosfolípids marcats i (ii) la interacció entre LacY amb els fosfolípids més llunyans o bulk s’ha investigat a través de làmines de lípid i proteïna sobre un suport, les quals s’han analitzat a partir de diversos modes de microscòpia de força atòmica (topografia, espectroscòpia de força i force-volume). En primer lloc, s’ha validat la preferència de LacY pels fosfolípids en fases fluïdes (Lα). A més, s’ha confirmat una composició lipídica entre la regió anulars i el bulk. Així, els fosfolípids bulk, considerats com a fosfolípids en fase Lα, tenen PG com a principal component, mentre que PE és el major component de la regió anular. Això sembla indicar una selectivitat entre LacY i els fosfolípids anulars. En segon lloc, s’ha descrit que la selectivitat de LacY per fosfolípid determinat a la regió anular està relacionada amb (i) càrrega neutra i (ii) curvatura espontània (C0) negativa. A més, D68 s’ha assenyalat com un aminoàcid important per la selectivitat de la proteïna envers els lípids anulars. Finalment, s’ha descrit una interacció recíproca entre LacY i els lípids bulk. Així, la presencia de la proteïna modifica la topografia i la nanomecànica del sistema lipídic, especialment de la fase Lα, i, alhora, la nanomecànica de la pròpia LacY varia segons la matriu lipídica que l’envolta. En conseqüència, la composició lipídica de la bicapa sembla determinar les forces que governen l’estreta interacció de LacY amb la membrana i, per tant, aquesta composició és decisiva per la correcta inserció i activitat de la proteïna.
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Setzler, Julia Christine [Verfasser], and W. [Akademischer Betreuer] Wenzel. "Theoretical and Experimental Investigations of the Interaction of Proteins and Nanoparticles with Biological Membranes / Julia Christine Setzler. Betreuer: W. Wenzel." Karlsruhe : KIT-Bibliothek, 2013. http://d-nb.info/1054989397/34.
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Cotta, Doné Stefania. "Nephrin - intracellular trafficking and podocyte maturation /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-411-2/.
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Rodriguez, Camargo Diana Carolina [Verfasser], Bernd [Akademischer Betreuer] [Gutachter] Reif, and Aphrodite [Gutachter] Kapurniotu. "Nuclear Magnetic Resonance Characterization of Aggregating Biological Systems and Membrane Proteins / Diana Carolina Rodriguez Camargo. Betreuer: Bernd Reif. Gutachter: Bernd Reif ; Aphrodite Kapurniotu." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/1105646866/34.
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Lucchesi, Pamela A. "Plasma Membrane Processes in Smooth Muscle: Characterization of Ca2+ Transport and Muscarinic Cholinergic Receptors: A Thesis." eScholarship@UMMS, 1989. https://escholarship.umassmed.edu/gsbs_diss/135.
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The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients.
The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability.
45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells.
As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3.
The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.
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Blodgett, David M. "Human Erythrocyte Glucose Transporter (GLUT1) Structure, Function, and Regulation: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/326.
Full textAbstract:
The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure.
A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions.
This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.
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Francisco, Rafael Neves. "Solubilization of membrane proteins using ionic liquids." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/21537.
Full textAbstract:
Mestrado em Biotecnologia, ramo biotecnologia industrial e ambientalThe main goal of this work consists on the study of the ability of ionic liquids (ILs) to extract membrane proteins from biological membranes while keeping their integrity in aqeuous solutions. Since typical surfactants are mainly used for this purpose, ILs are here investigated as a new class of extraction agents. For the evaluation of the ILs solvation ability power, four proteins were selected to be overexpressed in Escherichia coli and to be used as model proteins, namely the Outer Membrane Protein F (OmpF) and Outer Membrane Protein C (OmpC), that are β-barrel proteins, and two bacteriorhodopsins, Haloarcula marismortui (HmBRI) and Haloarcula walsbyi (HwBR), that α-helix proteins. In this work, the investigations carried out with OmpC failed during cloning and OmpF failed during the purification step. On the other hand, the two bacteriorhodopsins were expressed and purified successfully. Although some adjustments (mainly the His-tag) must be performed to improve the expression and the purification level, 1 mg/L of HmBRI and 0.1 mg/L of HwBR were purified. The protein HmBRI was then chosen as a model protein to test the extraction ability of several aqeuous solutions of ILs. Its interesting feature of producing solutions and pellets with a purple colour, and its specific absorbance at 552 nm, make of HmBRI an excellent model protein since it can be easily monotired by Vis-spectroscopy and analysis of its colour. Iimidazolium-, phosphonium-and cholinium-based ILs were investigated in several concentrations in aqueous solutions. None of the ILs studied revelaed to be able to extract HmBRI without denaturation. However, cholinium decanoate was able to extract a higher amount of protein from the biological membrane compared to the commercial detergent decyl maltoside. Mixtures using cholinium decanoate and decyl maltoside were then used to extract the HmBR altough no further improvements on the extraction were observed. Since HmBRI is reported as a good fusion tag for other membrane proteins, avian specific antibodies (polyclonal) were finally produced by immunizing quails to evaluate the performance of HmBRI as a fusion tag.
O objetivo principal deste trabalho consistiu no estudo da capacidade de líquidos iónicos para extrair proteínas de membrana de membranas biológicas, assim como em manter a sua integridade em solução aquosa. Para a avaliação da capacidade de extração, foram selecionadas quatro proteínas para sobreexpressar em Escherichia coli, nomeadamente Outer Membrane Protein F (OmpF) e Outer Membrane Protein C (OmpC), que são proteínas em barril-β, e duas bacteriorodopsinas, Haloarcula marismortui (HmBRI) e Haloarcula walsbyi (HwBR), que são compostas por hélices-α. Infelizmente a produção de OmpC falhou durante o passo de clonagem enquanto que a obtenção de OmpF falhou durante o passo de purificação. Por outro lado, as duas bacteriorodopsinas foram expressas e purificadas com sucesso. Embora alguns ajustes (principalmente ao nível da His-tag) devam ainda ser realizados para melhorar a expressão e a purificação num futuro próximo, por cada litro de cultura foram purificados 1 mg de HmBRI e 0,1 mg de HwBR. A proteína HmBRI foi finalmente escolhida como proteína modelo para testar a capacidade de extração e solubilização de vários líquidos iónicos. A sua característica interessante de produzir soluções e pellets com cor roxa, assim como a sua absorvência específica a 552 nm, faz da HmBRI uma excelente proteína modelo facilmente monitorizada.Os líquidos iónicos estudados são derivados de catiões imidazólio, fosfónio e colinio. De um modo geral, nenhum líquido iónico foi capaz de extrair HmBRI sem a desnaturar. No entanto, o decanoato de colinio foi capaz de extrair mais proteínas da membrana biológica em comparação com o detergente comercial, decilo maltosideo. Por fim, foram estudadas misturas de decanoato de colina e surfactante comercial para extrair, apesar de os resultados serem semelhantes ao quando utilizando apenas líquido iónico. Uma vez que a HmBRI é uma boa proteína de fusão para outras proteínas de membrana, foram produzidos anticorpos específicos de aves (policlonais) pela imunização de codornizes, sendo estes anticorpos posteriormente purificados a partir de gema de ovo. Estes anticorpos são muito úteis na investigação de HmBRI como tag fusão.
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Silva, Cleonice da. "Isolamento do transportador de trealose de Saccharomyces cerevisiae." Universidade de São Paulo, 1999. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-02102014-124552/.
Full textAbstract:
O gene AGT1 do locus mal1g, do sistema de transporte de maltose de S. cerevisiae, codifica uma proteína de 67 kDa, que tem 75% de similaridade e 58% de identidade com o transportador de maltose do locus MAL1. Sua expressão é ativada por genes reguladores constitutivos do sistema MAL, e é reprimida pela glicose. A cepa AP68-7A carrega o gene AGT1, e possivelmente o gene transportador MAL31, e transporta trealose ao final da primeira fase de crescimento anaeróbico. SDS-PAGE comparando proteínas de membranas de células reprimidas pela glicose (taxa de transporte <0,5 U/mg), com aquelas de células induzidas por α-metilglicosídeo (taxa de transporte de ~35 U/mg), verificou-se 2 bandas (PMs 57 e 66 kDa) exclusivas em membranas de células induzidas. As 2 bandas foram isoladas por três métodos diferentes (cromatografia de troca iônica, lavagens da membrana com tampão de alta força iônica e cromatografia de afinidade) e testadas para ligação de 14C-trealose. A ligação foi enriquecida ~3 X após a cromatografia de troca iônica. O transporte de trealose na cepa AP77-4C (que não tem nenhum dos genes transportadores dos loci MAL) foi recuperado após sua transformação com plasmídeo YEp366-AGT1. De membranas plasmáticas destas células (transporte de trealose ~25 U/mg) foram isoladas por cromatografia de afinidade, 2 bandas cujos PMs em SDS-PAGE são idênticos aos das proteínas isoladas das membranas da cepa AP68-7A. Estes resultados permitem concluir que o transportador de trealose de leveduras é codificado pelo gene AGT1.The AGT1 gene presente in the mal1g locus from S. cerevisiae maltose transport system encodes a 67 kDa protein which shares 75% similarity and 58% identity with the maltose transporter protein encoded in MAL6 locus. The expression of this gene is regulatory genes from MAL system and is repressed by glucose. The strain AP68-7A which harbors the AGT1 gene and probably the MAL31 transporter gene, expresses trehalose transport activity at the end of first anaerobic growth. The comparison from the SDS-PAGE of membrane proteins from glucose repressed cells (trehalose transport activity of <0.5 U/mg), and α-methylglucoside induced cells (trehalose transport activity of ~35 U/mg), revealed 2 bands (Mr 57 and 66 kDa) present only in the induced cells membranes. Those bands were isolated by 3 different methods (ionic exchange chromatography, high strength ionic washes and affinity chromatography, and tested for 14C-trehalose binding. Both bands bind trehalose and this activity was enriched about 3 times after the ionic exchange chromatography. The trehalose transport activity was recovered by strain AP77-4C, (which does not harbor any MAL transporter gene) after its transformation with a plasmid containing the AGT1 gene. From the membranes of these cells (trehalose transport activity of ~25 U/mg) 2 bands were isolated by affinity chromatography with similar Mrs to those isolated from AP68-7A strain. The results permit to conclude that the AGT1 gene encodes the yeast trehalose transporter.
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Lee, Kil Sun. "Biologia da proteína prion celular." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22082018-104148/.
Full textAbstract:
O prion celular (PrPc) é uma glicoproteína ligada à membrana plasmática por uma âncora de GPI (glycosylphosphatidylinositol). A sua isoforma anormal (PrPsc) é uma molécula infecciosa que causa várias doenças neurodegenerativas em mamíferos. A etiologia dessas doenças está associada a uma mudança conformacional pós-traducional de PrPc que ocorre após sua internalização (Prusiner, 1998). Na tentativa de desvendar as funções fisiológicas de PrPc, nosso grupo tem identificado e caracterizado as interações celulares que PrPc participa. A primeira delas é a interação entre PrPc e STI1 (Stress Inducible Protein 1). Essa interação transduz sinalização por cAMP e PKA levando a neuroproteção contra morte celular programada (Chiarini e cols, 2002; Zanata e cols, 2002). A segunda é a interação específica que existe entre PrPc e as proteínas da matriz extracelular, laminina e vitronectina, contribuindo para os processos neuronais, tais como crescimento, manutenção (Graner e cols., 2000 a e b) e regeneração dos neuritos (Hajj e cols., submetido), além da formação de memória de curta e longa duração (Coitinho e cols., submetido). Na primeira parte deste trabalho, procuramos investigar os genes regulados pelos sinais resultantes dessas interações e também pela remoção de PrPc usando a técnica de \"differential display\'\' RT-PCR. Na segunda parte do trabalho, caracterizamos que a interação PrPc - laminina é capaz de induzir uma sinalização transitória de cálcio, a qual ocorre mesmo na ausência de cálcio do meio extracelular. PrPc é uma molécula que cicla continuamente entre a membrana plasmática e os compartimentos intracelulares. Estudos recentes têm correlacionado o processo de internalização de PrPc com alguns dos seus papeis fisiológicos, tais como, homeostase de Cu2 + (Brown, 2001 ), interação com receptor de laminina (Gauczynski e cols, 2001) e até na conversão de PrPc para PrPsc (McKinley e cols, 1991; Arnold e cols, 1995). Portanto, na terceira parte deste trabalho, caracterizamos a localização e o tráfego celular de PrPc mostrando que PrPc está localizado na membrana plasmática e em compartimentos intracelulares e que trafega pelo Golgi, membrana plasmática, endossomos iniciais e de reciclagem. Foram mapeados ainda domínios na região amino-terminal responsáveis pela internalização de PrPc e na região carboxi-terminal como participantes da via secretora. Este trabalho contribuiu para o esclarecimento de alguns eventos biológicos relacionados à sinalização e ao tráfego de PrPc. Estes achados são de grande importância para a determinação das funções celulares de PrPc e ainda dos mecanismos envolvidos com as doenças relacionadas com esta molécula.The cellular prion protein (PrPc) is a glycoprotein anchored to the plasma membrane by GPI (Glycosyl-phosphatidylinositol). Its abnormal isoform (PrPsc) is the infectious protein responsible for several neurodegenerative diseases. The main etiology of the prion diseases is related to conformational changes in the PrPc molecule, which occur after its internalization (Prusiner, 1998). In order to elucidate the physiological functions of PrPc, our group identified and characterized interactions between PrPc and other cellular molecules. The first is the interaction between PrPc and STI 1 (Stress Inducible Protein 1). This interaction has an important role in the neuroprotection against apoptosis through cAMP and PKA signaling (Chiarini et al., 2002; Zanata et al., 2002). PrPc also interacts with proteins of the extracellular matrix such as laminin and vitronetin. These interactions contribute for neurite outgrowth, maintenance and regeneration (Graner et al., 2000 a and b; Hajj et al., submitted) and also in memory formation (Coitinho et al., submitted). In the first part of this work we have applied the differential dysplay RTPCR technique in order to identify genes that are regulated by PrPc - STI 1 interaction and also by the deletion of PrPc. In the second part we have demonstrated that PrPc-laminin interaction induces transient calcium signaling in neuronal cells, which occurs even in the absence of extracellular calcium. PrPc cycles continuously between the plasma membrane and intracellular compartments. This mechanism is associated with some of the physiological function of PrPc, such as Cu2+ homeostasis (Brown, 2001 ), interaction with laminin receptor (Gauczynski et al., 2001 ), and PrPc conversion into PrPsc (McKinley et al., 1991; Arnold et al., 1995). Thus, in the third part of this project, we have characterized the PrPc localization at the cell surface and in intracellular compartments. The protein trafficking through Golgi apparatus, plasma membrane, early and recycling endosomes was also defined. Moreover, we have determinated that N-terminus PrPc domain is responsible for its internalization while C-terminus participates in PrPc delivery. Therefore, this work has contributed to elucidate biological events related to the cell signaling and trafficking of PrPc, which are important for the characterization of PrPc physiological functions and to understand the pathological mechanisms related to this molecule.
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Fraser, Diane Patricia. "Theoretical studies of lipid-protein interactions in biological membranes." Thesis, University of Central Lancashire, 1987. http://clok.uclan.ac.uk/20009/.
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Monte Carlo simulations are used to investigate the conformational and orierjtational properties of lipids and proteins in a bilayer membrane. in the first instance, linear, hard-core tri-atomics are used to represent the two-dimensional projections of the lipid molecules. Studies on lipid only systems show that the average number of gauche rotations and the cross-sectional area of the lipids decrease with increasing density. There is no long range orientational order within the lipids but the local orieritational order increases with increasing density. No first order phase transitions are observed though a glassy solid is observed at high densities. The properties of the bulk lipid are unchanged upon the addition of protein molecules represented by hard discs of varying sizes. The nearest neighbour lipids are unchanged conformationally but are found to exhibit a high degree of orientational ordering around the proteins preferring to have the long axis of their projections parallel to the proteins surface. The degree of ordering increases with increas-ing density and decreasing curvature of the protein. The lateral pressure is almost independent of protein size or concentration if expressed as a function of the bulk lipid density. The hard-core of the lipids is softened to a site-site Lennard-Jones potential. The particles are found to cluster within the periodic cell used. There is a critical density below which the average number of gauche rotations and the cross-sectional area change little and the local orientational order increases. Above this density these properties are the same as for the hard-core systems. The structure, indicated by the radial distribution functions, is much greater than that for the hard-core systems and is almost independent of density. The invariance of the lipid conformations and the observed lipid-protein orientational order resolve the conflict that has arisen in the past regarding the presence or absence of an annulus of lipid around integral protein molecules. The different experimental methods are seen to examine different lipid properties.
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Rodríguez, Banqueri Arturo. "A random approach to stabilize a membrane transport protein for crystallization studies / Un enfoque aleatorio para estabilizar un transportador de membrana para estudios de cristalización." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/109040.
Full textAbstract:
X-ray crystallography is, now at days, one of the most powerful techniques to study proteins at the atomic level. Unfortunately, obtaining high quality crystals of membrane proteins for x-ray diffraction is a difficult task due to the hydrophobic nature of these proteins. The low stability in solution of these proteins and their tendency to form aggregates are the biggest problems during crystallization studies. One of the most common strategies to overcome these problems consists on working with functional mutants of these proteins. It has been reported that single point mutations of key residues (normally within transmembrane segments) leads to a remarkable increase in the stability of some membrane proteins after detergent solubilization and extraction from the membrane. In addition, a single mutation can stabilize a specific conformer of the protein, decreasing its heterogeneity in solution. Despite this, predicting what mutations are going to improve the stabilization of a protein is virtually impossible.
The main purpose of this thesis is to build up a medium-high throughput experimental protocol with the objective to generate and characterize random mutants of a membrane protein with more stability in detergent-solubilized solution and, therefore with a better probability to crystallize. The combination of random mutagenesis with rapid and sensitive screening protocols of protein expression and stability seems to be the best approach for this goal. The use of the green fluorescent protein (GFP) as reporter has enormously facilitated the studies of expression, purification and stability of a membrane protein. Also, with the aim of minimizing undesired effects of full-length GFP, we optimized an assay based on a split GFP to build and characterized the random mutants library.
Specifically we focus on SteT, a Bacillus subtillis transporter that exchanges L-threonine by L-serine. SteT is an excellent prokaryotic model (30% of amino acid identity) of the mammalian L-amino acid transporter (LAT) family. Genetic mutations of some LATs are the direct cause of two types of aminoaciduries. Moreover, a member of this family, LAT1, is overexpressed in tumor cells, although the physiological role of this is still unknown. Unfortunately, SteT wild type solubility and stability in detergent solutions is very low and completely incompatible with crystallization tests.
Our results suggest that random mutagenesis combined with the GFP split assay, appears to be an excellent strategy to build robustness in membrane proteins for structural studies. So far, using this strategy we found a mutant of SteT that currently is undergoing for crystallization screenings to study the structure and mechanism of mammalian LATs.La cristalografía de rayos X es, hoy en día, una de las técnicas más potentes para el estudio de las proteínas a nivel atómico. Desafortunadamente, la obtención de cristales de alta calidad de proteínas de membrana para la difracción de rayos X es un desafío debido a la naturaleza hidrofóbica de estas proteínas. La baja estabilidad en solución de estas proteínas y su tendencia a formar agregados son los mayores problemas durante los estudios de cristalización. Una de las estrategias más comunes para superar estos obstáculos consiste en trabajar con mutantes funcionales de estas proteínas. Se han publicado estudios sobre mutaciones en residuos clave en proteínas de membrana (normalmente dentro de los segmentos transmembrana) que conducen a un notable incremento de la estabilidad en solución, previa extracción de la membrana y solubilización en detergente. Además, una sola mutación puede estabilizar un confórmero específico de una proteína, disminuyendo su heterogeneidad en solución. A pesar de esto, predecir qué mutaciones van a mejorar la estabilidad de una proteína es prácticamente imposible. El principal objetivo de esta tesis es la construcción de un protocolo de alto rendimiento experimental con el objetivo de generar y caracterizar mutantes aleatorios de una proteína de membrana que presenten una estabilidad adecuada después de solubilizar la proteína en detergente y, por lo tanto, con mejores garantías de cristalizar. Para conseguir estos objetivos hemos combinado técnicas de mutaciones aleatorias con métodos de cribaje rápidos y sensibles. En este sentido, el uso de la proteína fluorescente verde (GFP) ha facilitado enormemente los estudios de expresión y purificación de proteínas de membrana. Con el objetivo de minimizar los efectos no deseados de la GFP, se creó y optimizó un ensayo basado en la complementación de la GFP (GFP split system) con un fin doble: seleccionar y caracterizar los componentes de la librería de mutantes aleatorios. Este protocolo se ha puesto a punto con SteT, un intercambiador de L-serina por L-treonina de Bacillus subtilis. SteT es un excelente modelo procariota (30% de identidad de aminoácidos) de la familia de transportadores de mamíferos de amino ácidos L (LAT). Mutaciones congénitas de algunos LATs son la causa directa de dos tipos de aminoacidurias. Además, un miembro de esta familia, LAT1, se sobreexpresa en células tumorales, aunque el papel fisiológico es aún desconocido. Desafortunadamente, SteT tiene una muy baja solubilidad junto a un gran inestabilidad en detergente, propiedades totalmente incompatibles con estudios de cristalización. Nuestros resultados indican que la mutagénesis aleatoria combinada con el ensayo basado en el “GFP split system”, es una estrategia excelente para aumentar la estabilidad de proteínas de membrana en estudios estructurales. Utilizando esta metodología hemos encontrado un mutante de SteT que actualmente está siendo cristalizado. Estos estudios serán clave para conocer mejor la estructura y el mecanismo de la familia de transportadores de mamífero LAT.
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Ying, Wang Li. "Study of the transport mechanism of the melibiose permease from Escherichia coli." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/377470.
Full textAbstract:
El transportador de melibiosa d'Escherichia coli (MelB) pot utilitzar l'energia electroquímica tant de H+, Na+ o Li+ per transportar la melibiosa a l'interior de les cèl·lules en contra del seu gradient de concentració. La MelB és una proteïna de 473 aminoàcids disposats en 12 hèlices transmembrana, amb els extrems N- i C-terminal situats al costat citoplàsmic. Mitjançant l'ús de mètodes espectroscòpics i bioquímics, hem analitzat el paper d'alguns aminoàcids en el bucle 7-8/final d'hèlix VII, que conté diversos aminoàcids aromàtics altament conservats, així com dos residus carregats negativament. Aplicant tècniques de mutagènesi, es van obtenir mutants individuals en què cada aminoàcid s'ha canviat a cisteïna excepte Ser-259, que també es va canviar a alanina. L'espectroscòpia de fluorescència va mostrar que els mutants dels aminoàcids conservats Tyr-256, Tyr-257, Phe-258 i Tyr-260 no uneixen substrats, i els espectres de diferència d'infrarojos de Y256C i Y260C van confirmar l’absència d’unió al substrat. Simulacions de dinàmica molecular van mostrar que aquests residus aromàtics formen part d'un bloc d’interaccions hidrofòbiques que jugaria un paper destacat en el mecanisme de transport. D'altra banda, els espectres de fluorescència i de diferència d’infraroig van demostrar que el mutant Cys del residu Asp-266 i del restant aminoàcid aromàtic del bucle 7-8 Phe-268 són capaços d'unir sodi i melibiosa en una forma semblant a la MelB nativa (Cless). Altres mutants de cisteïna (S259C, S259A, V261C, G263C, D264C, A265C i L267C) del bucle 7-8/final d'hèlix VII mostren una capacitat d'unió similar a Cless. Aquests resultats suggereixen que els aminoàcids conservats Tyr-256, Tyr-257, Phe-Tyr-258 i 260 tenen un paper estructural important en el mecanisme de transport de la MelB.The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H+, Na+ or Li+ to transport the melibiose to the cell interior against its concentration gradient. MelB is a protein of 473 amino acids arranged in 12 transmembrane helices, with the N- and C-terminus located in the cytoplasmic side. By using spectroscopic and biochemical methods, we have analyzed the role of some amino acids in the loop 7-8/end of helix VII, which contains several highly conserved aromatic amino acids as well as two negatively charged residues. Applying mutagenesis techniques, we obtained single mutants in which each amino acid has been changed to cysteine except Ser-259 also changed to alanine. Fluorescence spectroscopy showed that mutants of the conserved amino acids Tyr-256, Tyr-257, Phe-258 and Tyr-260 did not exhibit substrate binding, and the infrared difference spectra of Y256C and Y260C also showed no substrate binding. Molecular dynamics simulation experiments pointed out that these aromatic residues make part of a hydrophobic lock that would play a significant role in the transport mechanism. On the other hand, infrared difference and fluorescence spectra demonstrated that the Cys mutant of Asp-266 and the remaining aromatic amino acid of loop 7-8 Phe-268 are able to bind sodium and melibiose in a similar way as the wild type MelB (Cless). Other cysteine mutants (S259C, S259A, V261C, G263C, D264C, A265C and L267C) of the loop 7-8/end of helix VII show similar binding capacity as Cless. These results suggest that the conserved amino acids Tyr-256, Tyr-257, Phe-258 and Tyr-260 have an important structural role in the MelB transport mechanism.
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Silva, Marielle Garcia. "Análise proteômica de membrans de Paracoccidioides sp. durante privação de zinco." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/7643.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Paracoccidioides spp. are pathogenic fungi that causes paracoccidioidomycosis, an important systemic mycosis in Latin American countries. The success of infection depends on the pathogen ability to obtain essential micronutrients (metals) from the host. This process of nutrient uptake occurs through membrane associated transporters. The membranes are constituted of a lipid bilayer with associated proteins and are involved in different processes during the establishment of infection, such as transport of nutrients and homeostatic regulation. As zinc is a metal that plays an important role in the regulation of host-pathogen interaction and changes in this micronutrient homeostasis are implicated in the pathogenesis of infectious diseases, the aim of this study was to identify the membrane proteins expressed under conditions of zinc deprivation. NanoUPLC-MSE technique was employed in order to identify membrane proteins of yeast cells (Pb01) grown in chemically defined media in the presence and absence of zinc. Transmission electron microscopy was performed to confirm the sample enrichment with membranes. Subsequently, the samples were digested and analyzed by NanoUPLC-MSE. In silico analysis was performed to determine the location of 460 proteins identified in extracts of Pb01 grown in + Zn (control) and TPEN (treated) medium. Among the identified proteins, 141 were classified as belonging to membranes and of those, 120 proteins were repressed and 21 were induced during zinc deprivation. Among the 141 membrane proteins, 81 showed transmembrane domains and 9 were classified as membranes proteins with post-transcriptional modification. A total of 15 membrane proteins showed signal peptide. Analysis of the function of membrane proteins, allowed the description that phospholipid metabolism and cell integrity maintenance pathways were affected by zinc deprivation.
Paracoccidioides spp. são fungos patogênicos, causadores da paracoccidioidomicose, uma importante micose sistêmica nos países latino-americanos. O sucesso da infecção depende da capacidade do patógeno obter micronutrientes essenciais (metais) a partir do hospedeiro. Este processo de captação de nutrientes ocorre através de transportadores associados às membranas. As membranas são constituídas por uma bicamada lipídica com proteínas associadas e estão envolvidas em diferentes processos durante o estabelecimento da infecção, como transporte de nutrientes e regulação homeostática da célula. Como o zinco é um metal que desempenha um papel importante na regulação da interação patógeno-hospedeiro e distúrbios na homeostase deste micronutriente estão implicados na patogênese de doenças infecciosas, o objetivo deste estudo foi identificar as proteínas de membranas expressas em condições de privação de zinco. A técnica NanoUPLC-MSE foi empregada para identificar proteínas de membranas de células de levedura (Pb01) cultivadas em meio quimicamente definido na presença e ausência de zinco. Microscopia eletrônica de transmissão foi realizada para confirmar o enriquecimento da amostra com membranas. Posteriormente, as amostras foram digeridas e analisadas por NanoUPLC-MSE. Análises in silico foram realizadas para determinação da localização de 460 proteínas obtidas do extrato proteico de Pb01 cultivado em meio com zinco (controle) e TPEN (tratado). Do total de proteínas identificadas, 141 proteínas foram classificadas como pertencentes a membranas e destas, 120 proteínas foram reprimidas e 21 foram induzidas durante privação de zinco. Dentre as 141 proteínas de membranas, 81 apresentaram domínio transmembrana e 9 foram classificadas como sendo de membrana, em decorrência de modificação pós-traducional. Um total de 15 proteínas de membrana apresentou peptídeo sinal. Análises da função das proteínas de membranas permitiu a descrição de que o metabolismo de fosfolipídeos e a integridade celular foram afetados pela privação de zinco.
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Berry, Richard M. "Possible dynamic roles for the electrostatic force in biological membrane systems." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316866.
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Brandstaetter, Hemma. "Cellular function of the myosin1c motor protein in membrane trafficking." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609957.
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Nagpal, Kamalpreet. "A Tale of Two SNPS: Polymorphism Analysis of Toll-like Receptor (TLR) Adapter Proteins: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/540.
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The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins.
The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic.
Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population.
Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function.
This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents.
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Curcio, Juliana Santana de. "Proteínas da fração de membranas do fungo patogênico Paracoccidioides sp." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/7655.
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Made available in DSpace on 2017-08-07T15:54:46Z (GMT). No. of bitstreams: 2 Dissertação - Juliana Santana de Curcio - 2014.pdf: 2001377 bytes, checksum: 2f9ef22cd0a300cc93ee483fab0fb9c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-21
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Paracoccidioides spp. represents thermodimorphic fungi that cause paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. During the establishment of the infection, proteins located in cell membranes are responsible for many essential processes for pathogen survival as transport of nutrients, accession, signaling and energy production. Cell membranes are basically composed of a lipid bilayer with proteins associated. The proteins can be associated with the membrane through transmembrane domains, posttranslational added modifications or through electrostatic interactions. Therefore the knowledge of the constitution of the proteins of membranes is important to understand some processes developed by Paracoccidioides sp. for its growth and survival within the host. To this end, two techniques of separation and identification of proteins were employed. The first methodology used two-dimensional electrophoresis and mass spectrometry, where the extract of membranes proteins was obtained after steps of ultracentrifugation and the second methodology used liquid chromatography coupled with mass spectrometry, to this methodology three protocols were employed to obtain the extract of membranes proteins. The first methodology employed ultracentrifugation, and the second e third methodology used ultracentrifugation/sonication and ultracentrifugation with elevation of pH respectively. After these steps, the extract of membranes proteins was obtained through of ultracentrifugation and elevation of pH with sodium carbonate. Transmission Electron Microscopy (TEM) was performed to confirm the quality of the sample, a fraction enriched in cell membranes of Paracoccidioides sp.. Posteriorly the proteins obtained by standard methodology were subjected to tryptic digestion and analyzed by NanoUPLC-MSE.. In silico analyzes of the identified proteins were performed to determine the location and possible association of the proteins with the cell membranes. From total of proteins identified, one hundred thirty -three proteins were identified as belonging the cell membranes of Paracoccidioides sp.. Eighty-eight proteins showed at least one transmembrane domain and thirteen identified proteins showed a signal peptide at the N-terminus, beyond the transmembrane domain. Different forms of association of the proteins with membranes of Paracoccidioides sp. were detected, including domain transmembrane, GPI anchor, prenylation, myristoylation, palmitoylation. The most of the of integral membrane proteins identified in proteome were annotated in the genome of Paracoccidioides sp. Together these results show the establishment of an experimental protocol for the extraction of membranes proteins of Paracoccidioides spp., as well identify proteins from membrane fractions this fungus.
Paracoccidioides spp. representa um gênero de fungos termodimórficos, agentes etiológicos da paracoccidioidomicose, a micose sistêmica mais comum na América Latina. Durante o processo infeccioso proteínas localizadas em membranas celulares são responsáveis por muitos processos essenciais para a sobrevivência do patógeno, como transporte de nutrientes, adesão, sinalização e produção de energia. Basicamente membranas celulares são constituídas de uma bicamada lipídica com proteínas associadas. As proteínas de membranas associam-se a bicamada lipídica através de domínios transmembrana, por adição de uma modificação pós traducional e por meio de interações eletrostáticas. Portanto, o conhecimento da constituição proteica das membranas de Paracoccidioides sp. permite entender alguns dos processos desenvolvidos pelo fungo para sobrevivência dentro do hospedeiro. Desta forma o presente trabalho teve como objetivo à determinação da metodologia para obtenção de proteínas de membranas e a consequente descrição dessas proteínas. Para este fim, duas técnicas de separação e identificação de proteínas foram empregadas. A primeira metodologia baseava-se em eletroforese bidimensional e espectrometria de massas onde o extrato proteico de membranas foi obtido após duas etapas de ultracentrifugação e a segunda metodologia utilizava cromatografia líquida acoplada à espectrometria de massas, para esta metodologia três protocolos foram empregados no intuito de obter extratos proteicos de membranas. O primeiro protocolo emprega ultracentrifugação e o segundo e terceiro protocolo empregava ultracentrifugação/sonicação e ultracentrifugação combinado com elevação do pH respectivamente. Após as etapas de padronização do protocolo, o extrato proteico de membranas foi obtido através de ultracentrifugação e solubilização com carbonato de sódio em pH elevado. A microscopia eletrônica de transmissão (MET) foi realizada para confirmar a qualidade da amostra enriquecida com a fração de membranas de Paracoccidioides sp.. Posteriormente o extrato proteico de membranas proveniente da metodologia padronizada foi submetido à digestão tríptica e analisado por NanoUPLC-MSE. Análises in silico das proteínas identificadas foram realizadas a fim de se determinar a localização destas proteínas e possíveis associações com as membranas. Do total de proteínas identificadas cento e trinta e três proteínas foram classificadas como pertencentes a membranas celulares de Paracoccidioides sp., oitenta e oito proteínas apresentaram ao menos um domínio transmembrana e treze proteínas, além de domínio transmembrana apresentaram um peptídeo sinal na porção N-terminal da proteína. Diferentes formas de associação com as membranas de Paracoccidioides sp. foram identificadas neste trabalho, incluindo domínio transmembrana, âncora de GPI, prenilação, palmitoilação e miristoilação. A maioria das proteínas integrais de membrana identificadas no proteoma já foram anotadas no genoma deste fungo. Juntos estes resultados demonstram o estabelecimento de um protocolo experimental para a extração de proteínas de membranas de Paracoccidioides spp., bem como identificam as proteínas de frações de membranas do fungo.
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Fearnley, I. M. "Studies of mitochondrial membrane proteins." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380262.
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Zubiate, Fernando Alexis Gonzales. "Estudo de interações entre subunidades do exossomo e com outras proteínas celulares em Saccharomyces cerevisiae." Universidade de São Paulo, 2001. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23012019-094516/.
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Em Saccharomyces cerevisiae, Rrp43p é uma proteína que faz parte do exossomo, um complexo multiproteíco que atua no processamento de snoRNAs, snRNAs e rRNAs e na degradação de mRNA. O exossomo está envolvido nesses processos através de uma ação 3\'→5\' exonucleolítica. Este complexo é composto por onze subunidades em S. cerevisiae e cada uma dessas subunidades tem uma ação predominante nos diferentes processos em que o complexo participa. Por estar envolvido diretamente na maturação dos rRNAs e alguns snoRNAs e snRNAs, assim como na degradação de mRNAs, o exossomo tem um papel importante no controle de expressão gênica. Com o objetivo de entender melhor a função da subunidade do exossomo Rrp43p, e conseqüentemente do complexo, nas modificações do RNA, realizamos estudos de interação entre proteínas através do método de \"two hybrid\", que permite analisar interações in vivo entre duas proteínas, convertendo-se assim, em uma ferramenta importante nestes estudos. Utilizando Rrp43p como \" isca\" estudamos interações com outras proteínas expressas em Saccharomyces cerevisiae, na procura de proteínas envolvidas em alguns eventos de processamento de RNA, que pudessem ajudar a esclarecer em maior detalhe o papel do exossomo na célula. Também examinamos interações da Rrp43p com os demais componentes do exossomo para determinar a possível estrutura deste complexo. Os resultados obtidos demonstram que Rrp43p interage com somente uma outra subunidade do exossomo, Rrp46p. A força desta interação, quando quantificada através do nível de expressão de um gene repórter, é compatível com o fato dessas proteínas formarem parte de um complexo. Estes dados constituem resultados inéditos a respeito da interação entre subunidades do exossomo. Os resultados evidenciam também a interação entre Rrp43p e uma proteína com função ainda não caracterizada em levedura (aqui denominada 137p). Esta interação foi detectada através do sistema do duplo híbrido, e depois confirmada por co-imunoprecipitação. A determinação da função desta nova proteína poderá ampliar as ferramentas de estudo da função e controle de atividade de Rrp43p e do exossomo.In the yeast Saccharomyces cerevisiae, Rrp43p is one of the eleven subunits of the exosome, a complex involved in the processing of snoRNAs, snRNAs and rRNAs, and in mRNA degradation. The exosome participates in these processes through a 3\'-to-5\' exonucleolytic activity. Each of the eleven subunits is predominately active in one or few of the processes in which the complex takes part. Since the exosome is involved directly in rRNAs, and in some snRNAs and snoRNAs maturation, as well as in mRNA degradation, it plays an important role on the control of gene expression. Aiming to a better understanding of the Rrp43p subunit function on RNA processing, we started a screening for Rrp43p-interacting proteins through the yeast two hybrid system. In this study we expected to find proteins interacting with Rrp43p, which were involved in some aspects of RNA processing, and would improve the current knowledge on the exosome function. In order to obtain more information about the complex structure, we have also studied the interactions between Rrp43p and the other exosome subunits. The results shown here demonstrate that Rrp43p interacts with only one other exosome subunit, Rrp46p. These results can help elucidate the final exosome structure. We also found the interaction of Rrp43p with a protein of yet uncharacterized function (here named 137p). This interaction, identified in the two hybrid system, was also confirmed through co-immunoprecipitation analysis, and the study of 137p function might bring new insights on Rrp43p function and control.
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Taylor, Andrew Mark. "Biophysical studies on the human erythrocyte anion transporter, band 3." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360571.
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Sommer, Bernhard. "Characterisation of multi-protein complexes involved in membrane trafficking in Drosophila melanogaster." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614968.
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