Dissertations / Theses on the topic 'Ligand binding interactions'
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Wade, R. C. "Ligand-macromolecule interactions." Thesis, University of Oxford, 1988. http://ora.ox.ac.uk/objects/uuid:576ce119-6a93-4eb0-a7e4-1f2513736dbd.
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The optimisation of ligand-macromolecule interactions is fundamental to the design of therapeutic agents. The GRID method is a procedure for determining energetically favourable ligand binding sites on molecules of known structure using an empirical energy potential. In this thesis, it has been extended, tested, and then applied to the design of anti-influenza agents. In the GRID method, the energy of a hydrogen-bond is determined by a function which is dependent on the length of the hydrogen-bond, its orientation at the hydrogen-bond donor and acceptor atoms, and the chemical nature of these atoms. This function has been formulated in order to reproduce experimental observations of hydrogen-bond geometries. The reorientation of hydrogen atoms and lone-pair orbitals on the formation of hydrogen-bonds is calculated analytically. The experimentally observed water structures of crystals of four biological molecules have been used as model systems for testing the GRID method. It has been shown that the location of well-ordered waters can be predicted accurately. The ability of the GRID method to assist in the assignment of water sites during crystallographic refinement has been demonstrated. It has also been shown that waters in the active site of an enzyme may be both stabilized and displaced by a bound substrate. Ligands have been designed to block the highly conserved host cell receptor site of the influenza virus haemagglutinin in order to prevent the attachment of the virus to the host cells. The protein was mapped energetically by program GRID and specific ligand binding sites were identified. Ligands, which exploited these binding sites, were then designed using computer graphics and energy minimization techniques. Some of the designed ligands were peptides and these were synthesised and assayed. Preliminary results indicate that they may possess anti-influenza activity.
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Vestergaard, Henrik Tang. "Diversity in competitive ligand-receptor interactions : electrophysiological studies of ligand-receptor interactions at native and recombinant GABAA receptors /." Cph. : Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2003. http://www.dfh.dk/phd/defences/henriktangvestergaard.htm.
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Davies, Thomas Glanmor. "Protein-ligand interactions for the OppA system." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311012.
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McFail-Isom, Lori. "Effects of ligand binding, coordinate error and ion binding on nucleic acid structure and conformation." Diss., Georgia Institute of Technology, 1998. http://hdl.handle.net/1853/30735.
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Schechner-Resom, Martina Gabriele. "Ligand binding and molecular flexibility : Studies on DNA gyrase B." Université Louis Pasteur (Strasbourg) (1971-2008), 2005. http://www.theses.fr/2005STR1A001.
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L’ADN gyrase est une enzyme vitale pour la bactérie grâce à sa capacité de manipuler les molécules d’ADN dans la cellule vivante. Cette capacité fait de l’ADN gyrase une cible idéale pour des composés anti-infectieux. Dans ce travail, l’ADN gyrase a été étudié par des méthodes de modélisatoin moléculaire. Une approche de conception de ligands basée sur la structure a été entreprise sur le sous-domaine N-terminal de 24 kDa de l’ADN gyrase B (domaine GHKL). La flexibilité de deux boucles du site actif du domaine GHKL a été étudiée par des simulations de dynamiques moléculaires en présence de différents ligands. Dans une dernière partie, une analyse des modes normaux du dimère du domaine N-terminal de 43 kDa a été entrepriseDNA gyrase is a vital bacterial enzyme necessary for the handling of the large DNA molecules in the living cell. Therefore DNA gyrase is an ideal target enzyme for anti-infectious compounds. In this work DNA gyrase has been studied by molecular modelling methods. A computational structure-based ligand design approach has been carried out on the N-terminal 24 kDa subdomain of DNA gyrase B (GHKL domain). To further examine the flexibility of two active site loops, molecular dynamics simulations have been carried out on the GHKL domain in different ligand binding conditions. In a final part, normal mode analysis has been carried out on the dimer of the 43 kDa domain of DNA gyrase B
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Shimokhina, Natalia. "Dissecting contributions to binding thermodynamics in ligand-protein interactions." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445371.
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Lampinen, Milla. "AMPA receptor ligand-binding domain : site-directed mutagenesis study of ligand-receptor interactions." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/lampinen/.
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Zhou, Min. "Understanding non-covalent interactions : cooperativity in ligand binding and enzyme catalysis." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615013.
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Limson, Janice Leigh. "Electrochemical studies of metal-ligand interactions and of metal binding proteins." Thesis, Rhodes University, 1999. http://hdl.handle.net/10962/d1018239.
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Electrochemical methods were researched for the analysis of metals, proteins and the identification of metal binding proteins. Adsorptive cathodic stripping voltamrnetry for metal analysis combines the inherent sensitivity of electrochemical techniques with the specificity of ligands for the nonfaradaic preconcentration of analytes at the electrode. The utility of catechol, resorcinol, 4-methylcatechol and 4-t-butylcatechol as ligands was explored for the sensitive analysis of copper, bismuth, cadmium and lead on a mercury film glassy carbon electrode. Metal complexes of lead, copper and bismuth with resorcinol showed the largest increase in current with increase in metal concentration, whereas complexes of these metals with 4-t-butylcatechol showed the lowest current response. Cadmium showed the highest current responses with 4-methylcatechol. The four metals could be determined simultaneously in the presence of resorcinol, although considerable interference was observed between bismuth and copper. The electroanalysis of cysteine and cysteine containing proteins at carbon electrodes are impaired by slow electron transfer rates at carbon electrodes, exhibiting high overpotentials, greater than 1 V vs Ag! Agel. Metallophthalocyanines have been shown to promote the electrocatalysis of cysteine at lowered potentials. Chemical modification of electrodes with appropriate modifiers is a means of incorporating specificity into electroanalysis, with applications in electrocatalysis. A glassy carbon electrode was modified by electrodeposition of cobalt (II) tetrasulphophthalocyanine [Co(II)TSPct to produce a chemically modified glassy carbon electrode (CMGCE). The CoTSPc-CMGCE catalysed the oxidation of cysteine in the pH range 1 to 10. The significance of this electrode is an application for analysis of proteins at biological pH's. A biscyanoruthenium(II) phthalocyanine CMGCE catalysed the oxidation of cysteine at 0.43 V vs Ag/AgCl a significant lowering in the overpotential for the oxidation of cysteine. Metallothionein, a metal binding protein, is believed to be involved in metal homeostasis and detoxification in the peripheral organs of living systems. A method for the quantitative determination of this protein utilising its high cysteine content was presented. At pH 8.4 Tris-HCl buffer, and using a CoTSPc-CMGCE modified by electrodeposition of the modifier, the anodic peaks for the oxidation of metallothionein was observed at 0. 90 V vs Ag/ AgCI. Ferredoxin is a simple iron-sulphur protein. One tenth of its residues are cysteine. Ferredoxin is involved in simple electron transfer processes during photosynthesis and respiration. Electrochemical studies of spinach ferredoxin were conducted at a CoTSPc-CMGCE. Anodic currents for the oxidation of the cysteine fragment of ferredoxin was observed at 0.85 V vs Ag/AgCl in HEPES buffer at pH 7.4, representing a new method for analysis of this protein. Voltammetric studies of its ferric/ferrous transition have shown quasi-reversible waves atE~ -0.62 V vs Ag/AgCl only in the presence of promoters. At a CoTSPc-CMGCE, a cathodic wave attributed to the reduction of Fe(III)/Fe(II) was observed at Epc -0.34 V vs Ag/AgCl. This represents an alternative method for voltammetric studies of the ferric/ferrous transition at significantly lowered potentials. Melatonin, a pineal gland hormone functions m setting and entraining circadian rhythms and in neuroprotection as a free radical scavenger and general antioxidant. Using adsorptive cathodic stripping voltammetry, the binding affinities of melatonin, serotonin and tryptophan for metals, were measured. The results showed that the following metal complexes were formed: aluminium with melatonin, serotonin and tryptophan; cadmium with melatonin and tryptophan; copper with melatonin and serotonin; iron (III) with melatonin and serotonin; lead with melatonin, tryptophan and serotonin, zinc with melatonin and tryptophan and iron (II) with tryptophan. The studies suggest a further role for melatonin in the reduction of free radical generation and in metal detoxification and may explain the accumulation of aluminium in Alzheimer's disease.
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Greguric, Antun, University of Western Sydney, of Science Technology and Environment College, and of Science Food and Horticulture School. "The DNA binding interactions of Ru(II) polypyridyl complexes." THESIS_CSTE_SFH_Greguric_A.xml, 2002. http://handle.uws.edu.au:8081/1959.7/620.
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This thesis reports on the synthesis, characterisation, enantiomeric resolution, 1H NMR structural study and physical evaluation of a series of certain bidentate ligand metal complexes, where ‘L-L’ denotes the ancillary bidentate ligand and ‘intercalator’ indicates the intercalating bidentate ligand. The L-L series varies in size and shape. Results of many tests and projects conducted are explained in detail.Master of Science (Hons)
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Buch, Mundó Ignasi 1984. "Investigation of protein-ligand interactions using high-throughput all-atom molecular dynamics simulations." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/101407.
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Investigation of protein-ligand interactions has been a long-standing application for molecular dynamics (MD) simulations given its importance to drug design. However, relevant timescales for biomolecular motions are orders of magnitude longer than the commonly accessed simulation times. Adequate sampling of biomolecular phase-space has therefore been a major challenge in computational modeling that has limited its applicability. The primary objective for this thesis has been the brute-force simulation of costly protein-ligand binding modeling experiments on a large computing infrastructure. We have built and developed GPUGRID: a peta-scale distributed computing infrastructure for high-throughput MD simulations. We have used GPUGRID for the calculation of protein-ligand binding free energies as well as for the reconstruction of binding processes through unguided ligand binding simulations. The promising results presented herein, may have set the grounds for future applications of high-throughput MD simulations to drug discovery programs.
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Greguric, Antun. "The DNA binding interactions of Ru(II) polypyridyl complexes /." View thesis View thesis, 2002. http://library.uws.edu.au/adt-NUWS/public/adt-NUWS20030410.094714/index.html.
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Thesis (M. Sc.) (Hons.) -- University of Western Sydney, 2002.A thesis presented to the University of Western Sydney in partial fulfilment of the rquirements for the degree of Master of Science (Honours), February, 2002. Includes bibliographical references.
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Siu, Kit-man Phyllis. "Luminescent cyclometalated platinum(II) complexes : protein binding studies and biological applications /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B30575357.
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Fischer, Marcus. "Structural biophysics of ligand, fragment and water interactions with substrate binding protein SiaP." Thesis, University of York, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.542843.
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Wang, Yan. "Effects of glucocorticoid receptor binding on base excision repair at deoxyuridine in the glucocorticoid response element." Online access for everyone, 2006. http://www.dissertations.wsu.edu/Thesis/Summer2006/y%5Fwang%5F072106.pdf.
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Copenhaver, Ann Savena. "Photoelectron spectroscopy of supported metal-metal interactions." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184742.
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The bonding in a series of ligand-bridged metal dimer complexes has been characterized by He(I) and He(II) photoelectron spectroscopy and approximate molecular orbital calculations. Bridging ligands such as carbonyl, nitrosyl, methylene and pyrazolyl in the complexes [CpFe(NO)]₂, [Cp*Fe(NO)]₂, [CpRu(NO)]₂, [Cp*Co(CO)]₂, [CpFe(CO)₂]₂, [Cp*Fe(CO)₂]₂, [CpFe(CO)]₂-μCO-μCH₂, [Cp*Fe(CO)]₂-μCO-μCH₂, [CpFe(NO)]₂- μCh₂, [CpRu(NO)]₂-μCH₂, [CpCo(CO)]₂-μCH₂, [CpRh(CO)]₂-μCH₂, [Ir(pyrazolyl)(CO)₂]₂, [Ir(3-methylpyrazolyl)(CO)₂]₂ and [Ir(3,5-dimethylpyrazolyl)(CO)₂]₂ are investigated and their effects upon metal-metal interactions are surveyed. Due to the presence of two d⁷ or d⁸ late metal atoms per molecule, these complexes display many overlapping ionization bands in a narrow valence ionization region. Attention has been given to modelling the photoelectron single ionization with asymmetric and symmetric Gaussians. The overlapping ionizations are successfully represented in terms of the model bandshapes. Thermodynamic relationships between bond dissociation and photoelectron ionization energies are also investigated. With relationships of this type, trends in bond energies may be correlated with ionization energies. Ligand inductive and bonding effects as well as small changes in molecular geometry cause shifts in the metal-based ionizations, which aid chemical understanding and interpretation of the molecular orbital picture. By comparing a series of related metal dimers, the assignment of related ionizations in the photoelectron spectra becomes apparent. Changes in ligand π accepting ability and changes in metal and formal oxidation states are also probed. Addition information is provided by vibrational fine structure in Cp₂Os, [CpFe(NO)]₂, and [Cp*Co(CO)]₂ and spin-orbit splitting in Cp₂Os. The metal-ligand backbonding combinations are found to be the most stable interactions and are responsible for the stability of the metal dimers. Metal-metal interactions are found to be relatively unimportant. Ligands with stronger π accepting abilities allow for more stabilized supported metal dimer complexes.
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Brown, Jennifer Louise. "Investigation of the molecular interactions between an anti-peptide antibody and its ligand." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318221.
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Siu, Kit-man Phyllis, and 蕭潔敏. "Luminescent cyclometalated platinum(II) complexes: protein binding studies and biological applications." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B4501498X.
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Qian, Yi. "Flipping a MAGUK switch : complex domain interactions regulating ligand binding to the tumor suppressor Dlg /." view abstract or download file of text, 2006. http://proquest.umi.com/pqdweb?did=1251819311&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2006.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 68-71). Also available for download via the World Wide Web; free to University of Oregon users.
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Laine, Jennifer M. "Protein Ligand Interactions Probed by NMR: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/617.
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Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena.
In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d.
Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research.
This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.
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Wong, Kar-ho. "Luminescent cyclometalated platinum(II) and gold(III) complexes for molecular recognition and DNA binding studies /." Hong Kong : University of Hong Kong, 1999. http://sunzi.lib.hku.hk/hkuto/record.jsp?B20357874.
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Wong, Yung Hou. "Molecular basis of the opioid receptor-ligand interactions : the role of guanine nucleotide-binding proteins." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303263.
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Ghosh, Shohini. "Analysis of the binding interactions between peptides and the MHC class II protein I-A(d) /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/8541.
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Shinada, Nicolas. "Détection, caractérisation et comparaison des interactions protéine - ligand." Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCC090.
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Appréhender le mécanisme fin de la liaison d’un ligand sur sa protéine est un enjeu majeur de l’industrie pharmaceutique. L’objectif principale de cette thèse est d’améliorer la compréhension de ce mécanisme par l’étude des interactions moléculaires. A cette fin, une méthode de détection de contacts à grande échelle fut mise en place. Les premiers chapitres illustrent les différents types d’interaction recensés dans la littérature ainsi que les outils qui ont été développés au cours de cette thèse pour en permettre l’analyse. A l’aide des contacts générés par notre méthode, une analyse exhaustive a été réalisée sur un ensemble de complexe protéine - ligand caractérisant l’environnement d’interaction des atomes halogènes. Les résultats obtenus mettent en avant la complexité de la description des interactions dans un contexte protéine - ligand. Une analyse structurale des modes de liaison des ligands issus de la PDB a été effectuée pour évaluer de manière quantitative cette diversité. Un travail spécifique sur ka redondance importante des représentations de complexes protéine - ligand a été effectué amenant une stratégie innovante dédiée aux complexes protéine - ligand. L’interaction et la fonction d’une protéine étant très étroitement liée à sa structure, le comportement dynamique des structures hélicoïdales dans les protéines a été étudiée. L’utilisation d’une méthode de regroupement met en évidence une stabilité jusque-là peu soupçonnée des hélices les plus raresApprehending the binding mechanism in a protein – ligand complex is a major goal in pharmaceutical industry. The objective of this thesis was to improve the understanding of this mechanism through molecular interactions study. Consequently, a large-scale contact detection protocol was designed to achieve this goal. The first chapters highlight known interaction types observed in the literature and the resulting tools that were developed during this thesis. Using our dataset of intermolecular contacts, a comprehensive analysis underlines the intricacy of describing interaction patterns of halogen atoms in the protein-ligand context. Then, a structural comparison of ligand binding modes quantitatively assesses its diversity on the entire PDB dataset. Finally, protein function and interaction mechanism are strongly related to its structure. Using a clustering approach, dynamic behavior of helix structures was highlighted through transitional patterns and unsuspected stable conformations for rare helices
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Sit, Kei Chun. "Investigation of the binding interactions between insulin and its receptor." Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/87276/1/Kei_Sit_Thesis.pdf.
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This project investigated the interactions between insulin and its receptor. A combination of computational and experimental investigations resulted in the identification of four residues in non-canonical sites that, when mutated, had detrimental effects on insulin binding. An increased understanding of the binding mechanism will aid future research into diseases involving the insulin receptor and its relatives and could potentially lead to new therapeutic avenues to combat these health related issues.
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Esswein, Stefan Theo. "Probing protein-ligand interactions via solution phase hydrogen exchange mass spectrometry." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4638.
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Mass spectrometry is a versatile, sensitive and fast technique with which to probe biophysical properties in biological systems and one of the most important analytical tools in the multidisciplinary field of proteomics. The study of nativestate proteins and their complexes in the gas-phase is well established and direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are becoming increasingly popular as a tool for screening and determining quantitative information on protein-protein and protein-ligand interactions. However, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting purely gas-phase results. This thesis details modification and advancement of solution phase techniques devised by Gross et al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX (Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these interactions with regards to high throughput analysis. The first part of this thesis describes the different developmental stages of the devised HPLC-front ends and their optimisation with myoglobin and insulin. The successfully developed HPLC-front end in conjunction with PLIMSTEX and SUPREX and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)- cyclosporin A (CsA) system, followed by a test screen with potential CypA binding ligands. Dissociation constants (Kd’s) within one order of magnitude to reported values are determined. In the third part of this thesis the application of the devised ESI-SUPREX methodology has been applied to anterior gradient 2 (AGr2) and the factor H complement control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide and a synthetic pentasaccharide, respectively, resulting in thermodynamical data for these protein-ligand interactions. For the AGr2 system another dimension of investigation has been added by temperature controlling the devised ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures. The final part of this thesis describes the application of the ESI-SUPREX methodology to probe folding properties of CypA in the presence of the self expressed and purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have been emphasised along with the stabilisation of a denatured CypA species.
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Rhoad, Jonathan Sidney. "DNA-binding carbohydrates for coordination to a photoactive dirhodium complex and molecular dynamics studies of methyl furanosides evaluation of available force fields /." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1101315894.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xviii, 160 p.; also includes graphics Includes bibliographical references (p. 117-120). Available online via OhioLINK's ETD Center
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Bai, Liping. "The noncovalent binding of benzophenathridine alkaloids to double-stranded, bulged and G-quadruplex DNA." HKBU Institutional Repository, 2008. http://repository.hkbu.edu.hk/etd_ra/910.
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黃家豪 and Kar-ho Wong. "Luminescent cyclometalated platinum(II) and gold(III) complexes for molecular recognition and DNA binding studies." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31221919.
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Gonzalez, Walter G. "Protein-Ligand Interactions and Allosteric Regulation of Activity in DREAM Protein." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/2503.
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Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.
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Desaphy, Jérémy. "L'analyse structurale de complexes protéine/ligand et ses applications en chémogénomique." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-00997394.
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Comprendre les interactions réalisées entre un candidat médicament et sa protéine cible est un enjeu crucial pour orienter la recherche de nouvelles molécules. En effet, ce processus implique de nombreux paramètres qu'il est nécessaire d'analyser séparément pour mieux comprendre leurs effets.Nous proposons ici deux nouvelles approches observant les relations protéine/ligand. La première se concentre sur la comparaison de cavités formées par les sites de liaison pouvant accueillir une molécule. Cette méthode permet d'inférer la fonction d'une protéine mais surtout de prédire " l'accessibilité " d'un site de liaison pour un médicament. La seconde tactique se focalise sur la comparaison des interactions non-covalentes réalisées entre la protéine et le ligand afin d'améliorer la sélection de molécules potentiellement actives lors de criblages virtuels, et de rechercher de nouveaux fragments moléculaires, structuralement différents mais partageant le même mode d'interaction.
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Almlöf, Martin. "Computational Methods for Calculation of Ligand-Receptor Binding Affinities Involving Protein and Nucleic Acid Complexes." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7421.
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The ability to accurately predict binding free energies from computer simulations is an invaluable resource in understanding biochemical processes and drug action. Several methods based on microscopic molecular dynamics simulations exist, and in this thesis the validation, application, and development of the linear interaction energy (LIE) method is presented.
For a test case of several hydrophobic ligands binding to P450cam it is found that the LIE parameters do not change when simulations are performed with three different force fields. The nonpolar contribution to binding of these ligands is best reproduced with a constant offset and a previously determined scaling of the van der Waals interactions.
A new methodology for prediction of binding free energies of protein-protein complexes is investigated and found to give excellent agreement with experimental results. In order to reproduce the nonpolar contribution to binding, a different scaling of the van der Waals interactions is neccesary (compared to small ligand binding) and found to be, in part, due to an electrostatic preorganization effect not present when binding small ligands.
A new treatment of the electrostatic contribution to binding is also proposed. In this new scheme, the chemical makeup of the ligand determines the scaling of the electrostatic ligand interaction energies. These scaling factors are calibrated using the electrostatic contribution to hydration free energies and proposed to be applicable to ligand binding.
The issue of codon-anticodon recognition on the ribosome is adressed using LIE. The calculated binding free energies are in excellent agreement with experimental results, and further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with a ribosome loaded with the Phe UUU codon. The simulations also support the previously suggested roles of A1492, A1493, and G530 in the codon-anticodon recognition process.
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Beuming, Thijs. "Structure/function studies of membrane proteins : from molecular modeling and ligand binding to protein-protein interactions of the dopamine transporter /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1432803991&sid=3&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Prasannan, Charulata Bhaskaran. "Modulation of restriction enzyme PvuII activity by metal ion cofactors." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2009. http://etd.umsl.edu/r4461.
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Sahai, Michelle Asha. "Computational studies of ligand-water mediated interactions in ionotropic glutamate receptors." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b86d2f5a-3554-44c0-b985-5693241369ec.
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Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.
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Andersson, David. "Multivariate design of molecular docking experiments : An investigation of protein-ligand interactions." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-35736.
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To be able to make informed descicions regarding the research of new drug molecules (ligands), it is crucial to have access to information regarding the chemical interaction between the drug and its biological target (protein). Computer-based methods have a given role in drug research today and, by using methods such as molecular docking, it is possible to investigate the way in which ligands and proteins interact. Despite the acceleration in computer power experienced in the last decades many problems persist in modelling these complicated interactions. The main objective of this thesis was to investigate and improve molecular modelling methods aimed to estimate protein-ligand binding. In order to do so, we have utilised chemometric tools, e.g. design of experiments (DoE) and principal component analysis (PCA), in the field of molecular modelling. More specifically, molecular docking was investigated as a tool for reproduction of ligand poses in protein 3D structures and for virtual screening. Adjustable parameters in two docking software were varied using DoE and parameter settings were identified which lead to improved results. In an additional study, we explored the nature of ligand-binding cavities in proteins since they are important factors in protein-ligand interactions, especially in the prediction of the function of newly found proteins. We developed a strategy, comprising a new set of descriptors and PCA, to map proteins based on their cavity physicochemical properties. Finally, we applied our developed strategies to design a set of glycopeptides which were used to study autoimmune arthritis. A combination of docking and statistical molecular design, synthesis and biological evaluation led to new binders for two different class II MHC proteins and recognition by a panel of T-cell hybridomas. New and interesting SAR conclusions could be drawn and the results will serve as a basis for selection of peptides to include in in vivo studies.
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Cramer, Jonathan [Verfasser], and Gerhard [Akademischer Betreuer] Klebe. "Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin / Jonathan Cramer ; Betreuer: Gerhard Klebe." Marburg : Philipps-Universität Marburg, 2018. http://d-nb.info/1164156055/34.
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Ismail, Matthew Arif. "DNA-ligand interactions : a biophysical study of 9-hydroxyellipticine, Hoechst 33258 and a meso-substituted porphyrin derivative binding to DNA." Thesis, University of Warwick, 1998. http://wrap.warwick.ac.uk/4314/.
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The binding of 9-hydroxyellipticine (9-OHE), Hocchst-33258 (Hoechst) and trans-bis-(4- N-methylpyridiniumyl)diphenylporphyrin (t-112P) to DNA has been studied by means of spectroscopic techniques. The binding modes of t-H2P to calf thymus DNA (ct-DNA), poly[d(G-C)12 and poly[d(A-T)12 were dependent on ionic strength, ligand load on DNA and the base composition at the binding site. At low ionic strength, flow linear dichroism (LD) data suggested t-H2P binds to ct-DNA and poly[d(A-T)12 in an orientation consistent with groove binding and to poly[d(G-C)12 in an orientation consistent with partial intercalation. Interpretation of the spectroscopic data was facilitated by molecular modelling. At high ionic strength, circular dichroism (CD) and resonance light scattering (RLS) measurements indicated the formation of extended t-H2P aggregates on DNA. In addition, the approximate, average transition moment polarisations of t-H2P were assigned from stretched film LD data to facilitate interpretation of the flow LD data. The interaction of Hoechst with poly[d(G-C)12 and poly[d(A-T)12 was studied with CD, LD, RLS and fluorescence spectroscopy. The mode of Hoechst binding to poly[d(G-C)]2 was ligand load independent. The collective spectroscopic data suggested abinding mode in which part of the molecule intercalates and part lies extensively stacked in the poly[d(G-C)12 major groove. Hoechst binding to poly[d(A-T)12 was ligand load dependent. The molecule was observed to bind as dimers or small oligomers at the lower ligand loads studied, with extended aggregates being formed as the concentration of bound Hoechst was increased. LD measurements suggested a binding orientation inconsistent with minor groove binding thus it was concluded that the molecule stacked in the major groove. 9-OHE binding to ct-DNA, poly[d(G-C)12 and poly[d(A-T)12 was studied with LD, RLS and normal absorption spectroscopy. Binding by intercalation was shown to occur with each of the three DNAs. The intercalated mode was shown to be complemented at high ligand load by a stacked binding mode. LD data pertaining to the stacked mode were inconsistent with minor groove binding in which the 9-OHE plane lies at 45' to the helix axis, thus it was concluded that the stacking occurred in the major groove. The stacked binding mode occurred most readily on poly[d(G-C)12. The flow LD data were complemented with film LD measurements from which the approximate, average transition moment polarisations were assigned.
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Pipier, Angélique. "Etudes des G-quadruplexes : impact de la stabilisation par des ligands en tant qu'agents anti-cancéreux et identification des protéines associées régulant leur métabolisme." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30118.
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Les G-quadruplexes (ou G4) sont des structures non canoniques des acides nucléiques formées à partir de séquences riches en guanines. Les G4 sont des structures stables, présentes sur l'ensemble du génome et qui peuvent adopter différentes conformations. La formation des G4 peut réguler, de façon positive ou négative, différents processus cellulaires tels que la transcription, la réplication, les transactions des ARN et les mécanismes mitochondriaux. L'ensemble de ces processus nécessite le recrutement de protéines capables de moduler la formation de ces structures. Certaines protéines, telles que les hélicases BLM, WRN ou DHX36, sont capables de dérouler les G4 alors que d'autres, comme la nucléoline (NCL), se lient aux G4 et les stabilisent. Enfin, des molécules capables de stabiliser les G4 appelées ligands de G4, peuvent impacter divers processus dans lesquels sont impliqués les G4 ; en particulier, ils peuvent entrainer la répression de l'expression d'oncogènes et mener à de l'instabilité génomique. Ainsi, les ligands de G4 sont considérés comme de potentiels agents anti-cancéreux. Mes travaux de thèses s'articulent autour de plusieurs problématiques concernant les G4 : 1/ l'amélioration des ligands de G4 et leur caractérisation ; 2/ le décryptage des mécanismes induisant de l'instabilité génomique suite à la stabilisation des G4 par des ligands ; 3/ l'identification des protéines capables de se lier aux G4 (ou GBP pour " G4 Binding Proteins "). Par des expériences biochimiques et biophysiques, j'ai participé à la caractérisation de ligands dérivés de porphyrine. Dans le cas du ligand AuMA, j'ai montré une augmentation à la fois de la capacité de stabilisation des G4 et de la spécificité envers les G4, par rapport à d'autres molécules dérivées de porphyrine. Cette molécule représente donc un meilleur potentiel thérapeutique que le TMPyP4, ligand largement étudié, dont elle est dérivée. J'ai également étudié l'instabilité génomique due à la stabilisation des G4 grâce à l'utilisation du ligand pyridostatine et du ligand CX5461, actuellement en phase II d'un essai clinique. Ces ligands induisent des cassures double brin de l'ADN (ou CDB) dépendantes de la transcription par l'ARN polymérase II et partiellement dues à la pause transcriptionnelle. Les CDB sont initiées par l'activité des Topoisomérases II, enzymes impliquées dans la résolution des stress topologiques de l'ADN dus à la transcription et à la réplication. Ces résultats montrent le rôle important de la transcription dans l'induction de l'instabilité génomique et ouvrent de nouvelles pistes thérapeutiques, dans le traitement de cancers dans lesquels ces protéines sont surexprimées ou par la combinaison avec d'autres chimiothérapies telles que l'étoposide afin d'en augmenter le potentiel cytotoxique. J'ai étudié les protéines se liant aux G4 grâce à des structures contraintes, bloquées dans une conformation particulière, en mettant au point un protocole de détection des GBP par des expériences de "Pull-Down" suivie d'une analyse par spectrométrie de masse. Ces résultats, validés par la liaison aux G4 de protéines déjà identifiées et caractérisées telles que WRN, DHX36 ou encore CNBP, ont permis l'identification de 425 GBP. Ainsi, j'ai mis en évidence de nouvelles GBP impliquées dans divers processus cellulaires tels que la réplication, la réparation de l'ADN, la transcription et le métabolisme des ARN. De façon annexe, l'étude de la protéine CNBP dans un modèle animal a permis de montrer que la régulation des G4 in vivo impacte la transcription et le développement embryonnaire, renforçant le rôle des G4 dans des organismes vivants. Mes travaux contribuent à étendre les connaissances sur les G4 et leurs ligands, particulièrement celles portant sur les mécanismes d'action des G4 pendant la transcription, et ouvrent de nouvelles perspectives thérapeutiquesG-quadruplexes (or G4) are non-canonical structures of nucleic acid formed from guanine-rich sequences. G4 are stable structures, present throughout the genome and could be folded into different conformations. G4 formation can regulate, positively or negatively, different cellular processes such as transcription, replication, RNA transactions and mitochondrial mechanisms. All these processes require the recruitment of proteins able to modulate the formation of these structures. Indeed, some proteins, such as BLM, WRN or DHX36 helicases, are able to unwind G4 while others, like nucleolin (NCL), bind to and stabilize G4. Finally, G4 ligands, small molecules stabilizing G4, can impact various processes in which G4 are involved; in particular, they can cause repression of oncogene expression and lead to genomic instability. Thus, G4 ligands are considered to be potential anti-cancer agents. My thesis work focuses on several issues concerning G4: 1/ the improvement of G4 ligands and their characterization; 2/ the deciphering of the mechanisms inducing genomic instability following G4 stabilization by ligands; 3/ the identification of proteins able to bind to G4 (or GBPs for "G4 Binding Proteins"). Through biochemical and biophysical experiments, I have participated in the characterization of porphyrin-derived ligands. In the case of the AuMA ligand, I showed an increase in both G4 stabilization capacity and G4 specificity, compared to other porphyrin-derived molecules. This molecule therefore represents a better therapeutic potential than TMPyP4, a widely characterized ligand from which it is derived. I have also studied the genomic instability due to G4 stabilization using the pyridostatin ligand and the CX5461 ligand, currently in Phase II of a clinical trial. These ligands induce DNA double-strand breaks (or DSBs) dependent on transcription by RNA polymerase II and partly due to the transcriptional pausing. DSBs are initiated by the activity of Topoisomerases II, enzymes involved in the resolution of DNA topological stresses due to transcription and replication. These results show the significant role of transcription in the induction of genomic instability and open up new therapeutic approaches in the treatment of cancers in which these proteins are overexpressed or by combining them with other chemotherapies such as etoposide to increase their cytotoxic potential. I have studied G4-binding proteins using constrained structures, blocked in a particular conformation, by developing a protocol for the detection of GBPs through Pull-Down experiments followed by mass spectrometry analysis. These results, validated by the binding to G4 of proteins already identified and characterized such as WRN, DHX36 or CNBP, allow the identification of 425 GBP. Thus, I have highlighted new GBPs involved in various cellular processes such as replication, DNA repair, transcription and RNA metabolism. Aside, the study of CNBP protein in a zebrafish model has shown that the regulation of G4 in vivo affects transcription and embryonic development, reinforcing the role of G4 in whole living organisms. My work contributes to extend the knowledge of G4 and their ligands, particularly the mechanisms of action of G4 during transcription, and is opening up new therapeutic perspectives
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Tevis, Denise Susanne. "Heterocyclic Diamidines Induce Sequence Dependent Topological Changes in DNA; A Study Using Gel Electrophoresis." unrestricted, 2009. http://etd.gsu.edu/theses/available/etd-04162009-154105/.
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Thesis (M.S.)--Georgia State University, 2009.Title from file title page. W. David Wilson, committee chair; Stewart A. Allison, Kathryn B. Grant, committee members. College of Arts and Sciences.Description based on contents viewed July 22, 2009. Includes bibliographical references (p. 85-87).
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Desuzinges-Mandon, Elodie. "Rôle du domaine extracellulaire d’ABCG2 dans l’homéostasie des porphyrines." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10236/document.
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ABCG2 est un transporteur de la famille ABC impliqué dans le phénotype de résistance aux drogues développé par certaines cellules, par exemple les cellules cancéreuses. Ce transporteur a aussi un rôle physiologique de détoxication de composés endogènes, notamment les porphyrines, molécules indispensables mais qui présentent une toxicité potentielle. Cette toxicité nécessite une prise en charge particulière, évitant à ces composés d’être libres en solution. Dans ce contexte, nous avons fait l’hypothèse qu’ABCG2 pourrait participer à cette détoxication en limitant l’accumulation des porphyrines dans les cellules en les présentant à un partenaire extracellulaire. Nous montrons qu’ABCG2 transporte de l’hème ainsi que certains de ses dérivés et précurseurs et que ces porphyrines, contrairement aux autres substrats d’ABCG2, se fixent sur un domaine extracellulaire spécifique d’ABCG2, ECL3, composé d’environ 70 acides aminés. L’affinité d’ECL3 pour les porphyrines est de 0,5 à 3,5 μM, suffisamment affine pour permettre leur fixation après transport.Nous montrons aussi que l’albumine sérique humaine, impliquée dans la détoxication de l’hème, récupère les porphyrines fixées sur ECL3 par une interaction directe avec ABCG2. L’ensemble de ce travail a donc permis d’une part de mieux comprendre le rôle d’ABCG2 dans la régulation de l’homéostasie des porphyrines, notamment l’hème, et d’autre part, de façon originale, d’identifier le mécanisme moléculaire par lequel cette détoxication s’effectueABCG2 belongs to the ABC-transporter family, involved in drug resistance developed by cells, notably cancer cells. This transporter has also a physiological role of endobiotic detoxification, in particular porphyrins that are essential but potentially toxic molecules. This toxicity implies a specific handle, to avoid them to remain free in solution. In that context, we hypothesized that ABCG2 participate to this detoxification, limiting the intracellular porphyrin accumulation by presenting them to an extracellular partner. We show that ABCG2 transports heme and some of its derivatives and precursors. Interestingly, these porphyrins, unlike other ABCG2 (non-porphyric) substrates, can bind to an extracellular domain, specific of ABCG2, ECL3, 70 residues-long. ECL3 displays affinities for porphyrins in the range of 0.5 to 3.5 μM, high enough to allow their binding after transport. We also show that human serum albumin, implicated in heme detoxification, releases porphyrins bound to ECL3 by a direct interaction with ABCG2. This work established a better comprehension of ABCG2 role in porphyrin and in particular heme homeostasis regulation. In addition, our results contribute to elucidate part of the molecular mechanism by which such regulation is carried out
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Ruhayel, Rasha A. "Multinuclear platinum anticancer therapeutics : insights into their solution chemistry and DNA binding interactions from NMR spectroscopy and molecular modelling." University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2010. http://theses.library.uwa.edu.au/adt-WU2010.0021.
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In the 1980's, Nicholas Farrell developed a range of structurally distinct multinuclear Pt complexes that form long-range interstrand crosslinks (IXLs) in DNA. The dinuclear complex [{trans-PtCl2(NH3)}2-µ-(H2N(CH2)6NH2)]2+ (1,1/t,t) was the first of this series to show promising results, however, it was the trinuclear complex [{trans-PtCl2(NH3)}2-µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2]4+ (1,0,1/t,t,t or BBR3464) that was chosen for clinical trials based on significantly increased cytotoxicity compared to 1,1/t,t and cisplatin. Molecular biology experiments have shown that 1,1/t,t exclusively forms IXLs in DNA in the 5'¿ 5' direction, whilst 1,0,1/t,t,t can form IXLs in both the 5'¿5' and 3'¿3' directions. Previously, 2D [1H,15N] HSQC NMR has been used to study the formation of 5'5' 1,4GG IXLs. The formation of 3'3' 1,4GG IXLs have been studied as part of this thesis. More recently, Pt complexes such as [{transPtCl2(NH3)}2{H2N(CH2)6(NH2(CH2)2NH2)(CH2)6NH2}]4+ (1,1/t,t6,2,6) and [{transPtCl2(NH3)}2{H2N(CH2)6(NH2)(CH2)6NH2}]3+ (1,1/t,t6,6), where the charged central Pt moiety of 1,0,1/t,t,t is replaced by a polyamine linker, have been developed in the Farrell group and show increased potency compared to 1,0,1/t,t,t. The complex 1,1/t,t 6,2,6 is a lead candidate currently undergoing Phase I clinical trials. Prior to the work presented in this thesis, little was known about the aquation chemistry or kinetics of DNA binding of these novel complexes. Reported in Chapter 3 is the study of the formation of 3'3' 1,4GG IXLs by both 1,0,1/t,t,t and 1,1/t,t in the duplex 5' {d(TATACATGTATA)2} (3314XL) (pH 5.4, 298K). A combination of 1D 1H and 2D [1H, 15N] HSQC NMR experiments was used to directly compare the results with the stepwise formation of the 5'5' 1,4GG IXL with the previously studied duplex, 5' {d(ATATGTACATAT)2} (5514XL), under the same conditions. Preassociation as well as aquation were similar, however, differences were observed at the monofunctional binding step with evidence for numerous monofunctional adducts. Both reactions did not yield a single 3'3' 1,4GG IXL, rather several adducts that could not be characterised. Molecular dynamics simulations of the 3'3' 1,4GG IXLs showed highly distorted lesions that may have implication in cellular repair processes.
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Anderson, Julie Annette. "Highly accurate computational characterization of weak interactions in biologically relevant prototypes : from hydrogen bonding in the water trimer to Pi stacking in protein/ ligand binding /." Full text available from ProQuest UM Digital Dissertations, 2007. http://0-proquest.umi.com.umiss.lib.olemiss.edu/pqdweb?index=0&did=1801490551&SrchMode=1&sid=19&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1258651706&clientId=22256.
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Thesis (Ph.D.)--University of Mississippi, 2007.Typescript. Vita. Major professor: Gregory S. Tschumper "May 2007." Includes bibliographical references (leaves [103]-121). Also available online via ProQuest to authorized users.
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Hemmingsson, Lovisa, and Johan Klasén. "In vitro studies of Thiopurine S-Methyltransferase: Ligand binding interactions and development of a new enzymatic activity assay for TPMTwt, TPMT*6 and TPMT*8." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-119652.
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Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity. In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX. Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.
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Bush, Derek B. "A Molecular Simulation Study of Antibody-Antigen Interactions on Surfaces for the Rational Design of Next-Generation Antibody Microarrays." BYU ScholarsArchive, 2017. https://scholarsarchive.byu.edu/etd/6631.
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Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.
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Penkler, David Lawrence. "In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1018938.
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The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
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Howard, Warren A. "Synthesis and characterisation of platinum(II) and ruthenium(II) polyamide conjugates." View thesis, 2008. http://handle.uws.edu.au:8081/1959.7/43899.
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Thesis (Ph.D.)--University of Western Sydney, 2008.A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.
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Wang, Ying-Hui. "Molecular interaction of zinc finger domain : study of androgen receptor DNA binding domain and SCA7 domain of Ataxin7 by NMR." Strasbourg, 2010. http://www.theses.fr/2010STRA6018.
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La voie de signalisation du récepteur des androgènes (AR) est impliquée dans la progression du cancer de la prostate, et il a été montré que des mutations dans ce domaine étaient responsables de l'activation constitutive des gènes placés sous le contrôle des hormones androgènes. Une de ces mutations transforme un résidu thréonine du DBD en alanine (T575A). Des expériences permettant de mesurer l'activité de transcription ont permis à l'équipe du Dr. Ceraline à l'IRCAD de montrer que la mutation T575A induit un changement de spécificité du récepteur. Alors que l'activité de promoteurs placés sous le contrôle d'éléments de réponse spécifique de AR diminue, celle des promoteurs placés sous le contrôle d'éléments non spécifique augmente. Ce changement de spécificité est corrélé à une modification de l'affinité du récepteur pour les éléments de réponse spécifiques et non spécifiques. Afin de comprendre le mécanisme de cette "reprogrammation" à l'échelle moléculaire, l'étude structurale des domaines DBD des récepteurs sauvage et muté a été entreprise par RMN. La comparaison des deux structures en solution a montré que la mutation n'altère pas le repliement du domaine et donc que la différence de reconnaissance des éléments de réponse n'est pas liée directement à la structure tridimensionnelle du domaine. Nous avons ensuite cherché à déterminer si l'altération de la fonction n'était pas due à une différence de dynamique de la chaîne peptidique. Afin d'étudier les mouvements moléculaires le long de la chaîne, des mesures de relaxation hétéronucléaire ont été effectuées et ont montré également une grande similarité dans le comportement dynamique des deux domaines, à l'exception d'une région située dans le premier doigt de zinc à proximité d'une histidine (H570), qui est conservée dans l'ensemble de la famille des domaines DBD des récepteurs nucléaires. Cette différence nous a conduit à mesurer, par RMN, le pKa de cette histidine pour les deux protéines. Nous avons ainsi montré que la mutation T575A induit une diminution de 0,5 unité de pH par rapport à la même histidine dans le domaine sauvage. L'analyse de la structure a permis de montrer que cette différence de pKa est liée à la perte d'une interaction entre le groupe hydroxyle de la thréonine 575 et le cycle imidazole de l'histidine. L'effet de la mutation sur le mécanisme de reconnaissance s'explique donc par un effet indirect dans lequel un acide aminé situé à distance de la région d'interaction modifie la surface électrostatique du domaine DBD. L'effet de la charge positive en position 570 sur la spécificité de reconnaissance de l'élément de réponse a ensuite été étudiée en construisant plusieurs mutants portant ou non une charge à cette position (mutants H570R et H570A). Ces études ont permis de confirmer l'importance de cette charge et l'ensemble de nos travaux fournissent un éclairage inédit sur les mécanismes de reconnaissance de l'ADN par les récepteurs nucléaires. .The androgen receptor (AR) is a ligand-activated transcriptional factor and a member of the nuclear receptor super family. AR shares a common structural and functional architecture with other members of nuclear receptors. The DNA binding domain of AR (ARDBD) binds to specific response elements as a homodimer. In the clinic, certain mutations in AR are associated with the progression of prostate cancer and have consequences for the treatment of patients with advanced prostate cancer. Previous studies showed that the mutation T575A, locating in the DNA binding domain, enhances the transcriptional activity regulated by full-length AR on promoters containing the non-specific response element compared to the wild type domain does not. These differences prompted us to study the molecular mechanism of ARDBD wild type and the T575A mutant. Structures of ARDBD wild type and T575A mutant revealed high similarity. However, dynamic behavior showed distinct differences between wild type and T575A mutant domains. The protonation state of H570 in ARDBD was found to be differed by the mutation. This loss of charge of H570 results in changes in transcriptional activity of AR. .
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Benkestock, Kurt. "Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery." Doctoral thesis, KTH, Analytisk kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4730.
Full textAbstract:
Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.QC 20100705
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Opuu, Vaitea. "Computational design of proteins and enzymes." Thesis, Institut polytechnique de Paris, 2020. http://www.theses.fr/2020IPPAX081.
Full textAbstract:
Nous proposons un ensemble de méthodes pour la conception de systèmes moléculaires. Notre stratégie consiste à utiliser comme modèle des machines naturellement optimisées, les protéines. Les protéines peuvent être des briques structurales, des transporteurs d'informations ou des catalyseurs chimiques. Nous utilisons ici des approches computationnelles, complémentaires aux voies expérimentales, pour concevoir de tels systèmes.Nous avons d'abord entièrement redessiné un domaine PDZ impliqué dans des voies métaboliques. Nous utilisons une approche physics-based basée sur la mécanique moléculaire, un modèle de solvant implicite et un échantillonnage Monte Carlo. Parmi plusieurs milliers de variants prédits pour adopter le repliement PDZ, trois ont été sélectionnés et montrent un repliement correct. Deux ont une affinité détectable pour les ligands peptidiques naturels.Nous avons ensuite re-dessiné le site actif de l'enzyme méthionyl-ARNt synthétase (MetRS). En utilisant un algorithme de type Monte Carlo adaptatif, nous avons sélectionné des variants pour l'affinité MetRS/méthionine (Met). Sur 17 variants testés expérimentalement, 17 sont actifs. La méthode a été ensuite appliquée à l'état de transition pour sélectionner des variants directement sur leur efficacité catalytique.Nous avons étudié la possibilité de modifier la MetRS pour étendre son activitéaux acides aminés β, afin d'étendre le code génétique. Ces acides aminésnon-naturels permettraient d'enrichir le répertoire structural des protéines. 20variants MetRS obtenus à partir de prédictions d'affinité MetRS/β-Met ont ététestés. Aucun n'augmente l'activité mais trois ont amélioré la sélectivité enfaveur de la β-Met. Nous avons implémenté une méthode de sélection de positionsd'intérêt et production de variants pour β-Met et β-Val. Une vingtaine deprédictions sont en cours de tests expérimentaux.Enfin, la modification de protéines in vivo pose la question de leur dérive génétique. Nous introduisons ici une méthode de conception de paires de gènes chevauchants pour des domaines PDZ. Ce codage permettrait de limiter la dérive génétique. Nous avons produit près de 2000 paires de domaines PDZ au codage chevauchant, dont une a été validées par 2 microsecondes de dynamique moléculaire. Des tests expérimentaux sont en coursWe propose a set of methods to design molecular systems. We start from naturally optimized components, namely proteins. Proteins can act as structural components, information transporters, or catalysts. We use computational methods to complement experiments and design protein systems.First, we fully redesigned a PDZ domain involved in metabolic pathways. We used a physics-based approach combining molecular mechanics, continuum electrostatics, and Monte Carlo sampling. Thousands of variants predicted to adopt the PDZ fold were selected. Three were validated experimentally. Two showed binding of the natural peptide ligand.Next, we redesigned the active site of the methionyl-tRNA synthetase enzyme (MetRS). We used an adaptive Monte Carlo method to select variants for methionine (Met) binding. Out of 17 predicted variants that were tested experimentally, 17 were found to be active. We extended the method to transition state binding to select mutants directly according to their catalytic power.We redesigned the MetRS binding site to obtain activity towards two β-amino acids, in order to expand the genetic code. These unnatural amino acids can enhance the structural repertoire of proteins. 20 predicted mutants were tested. Although none had increased β-Met activity, three had a gain in selectivity for β-Met. We then implemented a method to select optimal positions for design and applied it to β-Met and β-Val. Around 20 variants are being experimental tested.Finally, in vivo protein modifications raise the question of their eventual drift away from the original design. We introduce here a design approach for overlapping genes coding PDZ domains. This overlap would reduce genetic drift and provide bio-confinement. We computationally produced almost 2000 pairs of overlapping PDZ domains. One was validated by 2 microsecond molecular dynamic simulations. Experiments are underway