Rozprawy doktorskie: „Protein chemistry”

1

Slavoff, Sarah Ann. "Enzyme-mediated labeling of proteins and protein-protein interactions in vitro and in living cells". Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/62060.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
The E. coli biotin ligase enzyme, BirA, has been previously used by the Ting research group for site-specific labeling of peptide-tagged cell surface proteins. We sought to expand the utility of biotin ligase-mediated labeling to functional group handles, including azides and alkynes, for bio-orthogonal chemistry. Since the BirA and its point mutants were unable to ligate these probes to an acceptor peptide, we screened biotin ligases from multiple species to identify more permissive enzymes. We determined that the Pyrococcus horikoshii biotin ligase utilizes an azide-bearing biotin analog and that the Saccharomyces cerevisiae biotin ligase can utilize an alkyne-functionalized biotin analog. We subsequently demonstrated that the azidefunctionalized biotin analog can be derivatized with a phosphine probe via the Staudinger ligation. We next turned to the goal of delivering quantum dots to the cytosol of living cells, which in the future may permit intracellular single-molecule imaging. We investigated viral methods of delivery, but found that our protocol caused quantum dots to be trapped in endocytic vesicles. We then validated previous reports that the pore-forming toxin streptolysin 0 be used to deliver quantum dots to the cytosol of living cells. Lipoic acid ligase, or LpIA, has been previously applied to site-specific protein labeling of peptide-tagged proteins using small molecule probes including lipoic acid and coumarin fluorophores. We utilized LpIA and its substrate, the LAP peptide, to create sensors for proteinprotein interactions. If LpIA is fused to one protein and LAP is fused to another, only when the two proteins interact do LpIA and LAP come into proximity, allowing probe ligation onto the peptide to occur as a readout of the interaction. We demonstrate that proximity-dependent coumarin ligation detects protein-protein interactions in living mammalian cells with extremely low background, a signal-to-background ratio of at least 5:1, and sufficiently fast kinetics to label interactions with a half-life of at least 1 minute. The reporter quantitatively responds to subpopulations of interacting proteins, allowing dissociation constants to be measured. Coumarin fluorescence accurately reports the subcellular localization of the interaction under study. Finally, we applied proximity-dependent coumarin ligation to imaging of the interaction of PSD-95 and neuroligin-1, two proteins involved in synaptic maturation, in neurons.
by Sarah Ann Slavoff.
Ph.D.

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2

Bhat, Venugopal T. "Protein-directed dynamic combinatorial chemistry". Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/8758.

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Dynamic combinatorial chemistry (DCC) is a novel approach to medicinal chemistry which integrates the synthesis and screening of small molecule libraries into a single step. The concept uses reversible chemical reactions to present a dynamic library of candidate structures to a template which selects and removes the best binder from equilibrium. Using this evolutionary process with a biopolymer template, such as a protein, leads to the protein directing the synthesis of its own best ligand. Biological DCC applications are extremely challenging since the thermodynamic criterion of reversibility has to be met under physiological conditions to ensure stability of the biomolecular template. The list of reversible reactions satisfying these stringent criteria is limited and is a major constraint on achieving both reaction and structural diversity in adaptive dynamic libraries. This thesis reports the development of a catalysed version of acylhydrazone dynamic libraries which are truly adaptive under protein-friendly conditions. In the presence of aniline as a trans-imination catalyst, acylhydrazone dynamic combinatorial libraries equilibrate rapidly at pH 6.2 and are switched off by an increase in pH. We designed acylhydrazone libraries targeting the enzyme superfamily Glutathione-S-Transferase (GST) using a scaffold aldehyde, 4-chloro-3-nitrobenzaldehyde, which is structurally related to a known GST substrate chlorodinitrobenzene. On interfacing these dynamic libraries with two different GST enzymes (SjGST from the helminth worm Schistosoma japonicum and hGSTP1-1, a human isoform and an important oncology drug target) we observed isoformselective amplification effects with two different acylhydrazones selected by the proteins. To explore the potential of anchoring in our DCC methodology we conjugated the endogenous GST ligand, glutathione (GSH) onto the scaffold chloronitrobenzaldehyde. The GSH recognition motif acts as an anchor and allows us to explore the hydrophobic binding site of the enzyme in a fragment-based approach. The presence of the glutathione moiety led to increased solubility of the library members and a DCC experiment with the enzymes led to the selection of conjugate hydrazones with significant binding ability. Multi-level dynamic libraries use multiple exchange processes in the same system to increase their accessible structural diversity. These exchange reactions may be orthogonal, where the different chemistries can be activated or deactivated independently of each other, or simultaneous, where all the processes are dynamic and crossover under the same conditions. Together, these interacting molecular networks provide an exciting experimental approach to the emerging field of systems chemistry. We demonstrate that two reversible reactions, conjugate addition of thiols to enones and hydrazone formation, are fully compatible and orthogonal to one another in a single dynamic library. Hydrazone exchange takes place at acidic pH, while conjugate addition operates at basic pH. Simple pH change can be used to switch between each process and establish two channels of reactivity.

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3

Maset, Fabio. "Protein Chemistry and Molecular Medicine". Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3422744.

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Proteomics involves the systematic study of proteins in order to provide a comprehensive view of the structure, function and regulation of biological systems. Advances in instrumentation and methodologies have fueled an expansion of the scope of biological studies from simple biochemical analysis of single proteins to measurements of complex protein mixtures. Proteomics is rapidly becoming an essential component of biological research. Coupled with advances in bioinformatics, this approach to comprehensively describing biological systems will undoubtedly have a major impact on our understanding of the phenotypes of both normal and diseased cells. Initially, proteomics was focused on the generation of protein maps using two-dimensional polyacrylamide gel electrophoresis. Protein expression, or the quantitative measurement of the global levels of proteins, may still be done with two-dimensional gels, however, mass spectrometry has been incorporated to increase sensitivity, specificity and to provide results in a high-throughput format. Mass spectrometry applied to proteins offers many advantages: in addition to calculating the molecular weight with high accuracy, this technique allows to analyze and characterize the amino acid sequence. It can also be used in the study of post-translational modifications and to monitor the formation of complexes in solution. Finally it can be applied with different purposes such as the conformational analysis, analysis of the kinetics of refolding and studies on the catalytic activities of proteins. During my Ph.D. course my efforts have been mainly devoted on the use of this technique in combination with methods of protein chemistry such as the one- and two-dimensional electrophoresis, differents liquid chromatographies, solid phase peptide synthesis and use of proteolytic enzymes. Herein reported in my Ph.D. Thesis, the different treated subjects are divided into independent chapters each containing a single case study. Briefly in chapter 2 it has been proposed the study on protease nexin-1 (PN-1), the main inhibitor of thrombin in the brain, aimed at clarifying the role of the carbohydrate portion on the conformation, stability and function, through studies on recombinant protein produced in E.coli. In chapter 3 it has been showed the work on purification and chemical characterization, including de novo identification of amino acid sequence, of a similar inhibitor of phospholipase A2 extracted from the Python sebae serum, which demonstrated an effect cytotoxic pro-apoptotic and that could be exploited for the development of new anticancer strategies. In chapter 4 it has been reported the molecular dynamics that lead to the development of primary hyperoxaluria type I by studying the G41R mutant enzyme alanine: glyoxylate aminotransferase (AGT). In particular, were investigated the mechanisms leading to G41R be more susceptible to degradation and aggregation than wild type protein. Finally, chapter 5 deals with the effect of oxidative stress on the metabolism of von Willebrand Factor (VWF). The von Willebrand Factor is a very complex plasma glycoprotein whose dimensions help to regulate the hemostatic balance. Specifically, in the study was observed as the oxidation of a methionine residue located in the A2 domain of glycoprotein prevents proteolytic cleavage by ADAMTS-13, while not going to influence or, in some cases, promote proteolysis of VWF by proteases released from polymorphonuclear leukocytes in inflammatory conditions.
La proteomica riguarda lo studio sistematico delle proteine al fine di fornire una visione completa della funzione, della struttura e della regolazione dei sistemi biologici. I progressi avvenuti negli ultimi decenni, sia per quanto riguarda la strumentazione sia le metodologie utilizzate, hanno permesso di ampliare il campo di studi biologici passando dall’analisi di proteine purificate all’analisi di miscele complesse. La proteomica sta rapidamente diventando una componente essenziale della ricerca biologica ed associato ai progressi della bioinformatica, questo approccio alla descrizione dei sistemi biologici avrà indubbiamente un impatto notevole sulla nostra comprensione dei fenotipi sia delle cellule normali e malate. Inizialmente la proteomica era focalizzata principalmente sulla generazione di mappe proteiche bidimensionali utilizzando elettroforesi su gel di poliacrilammide. La verifica dell’espressione o la misurazione quantitativa dei livelli globali di proteine può ancora essere fatta sulla base dei gel bidimensionali, tuttavia oramai questi compiti sono affidati alla spettrometria di massa la quale può contare su di un’elevata sensibilità e specificità. La spettrometria di massa applicata alle proteine offre molti vantaggi: oltre a calcolare il peso molecolare con elevata precisione, questa tecnica permette di analizzare e caratterizzare la sequenza aminoacidica. Può anche essere utilizzata nello studio delle modificazioni post-traduzionali e per monitorare la formazione di complessi in soluzione. Infine può essere applicata con differenti scopi, quali l'analisi conformazionale, l'analisi della cinetica di ripiegamento e di studi sulle attività catalitiche delle proteine. Durante il dottorato di ricerca la mia attenzione è stata focalizzata soprattutto sull’utilizzo di tale tecnica abbinata a metodologie di chimica delle proteine quali ad esempio l’elettroforesi mono e bidimensionali, differenti cromatografie in fase liquida, la sintesi peptidica in fase solida e l’utilizzo di proteasi enzimatiche. In particolare in questa Tesi di Dottorato gli argomenti di studio sono stati trattati singolarmente, distinguendo i principali progetti in cui sono stato coinvolto in capitoli indipendenti. Brevemente, nel capitolo 2 è proposto lo studio di protease nexin-1 (PN-1), il principale inibitore della trombina a livello cerebrale, volto a chiarire la funzione della porzione glucidica sulla conformazione, stabilità e funzione della proteina mediante lo studio della proteina ricombinante prodotta in E. coli. Nel capitolo 3 è riportato il lavoro concernente la purificazione e la caratterizzazione chimica, in particolare dell’identificazione de novo della sequenza amminoacidica, di un analogo dell’inibitore della fosfolipasi A2 estratto dal siero di Python sebae, il quale ha dimostrato di possedere un effetto citotossico pro-apoptotico e che potrebbe essere sfruttato per lo sviluppo di nuove strategie antitumorali. Nel capitolo 4 l’attenzione è stata concentrata a chiarire le dinamiche molecolari che portano allo sviluppo di iperossaluria primaria di tipo I mediante lo studio del mutante G41R dell’enzima alanina:gliossilato amminotransferasi (AGT) analizzando in particolar modo i meccanismi che portano G41R ad essere maggiormente soggetto a degradazione e aggregazione rispetto alla proteina WT. Infine, il capitolo 5 tratta dell’effetto dello stress ossidativo sul metabolismo del fattore di von Willebrand (VWF). Il fattore di von Willebrand è una glicoproteina plasmatica estremamente complessa le cui dimensioni contribuiscono a regolare l’equilibrio emostatico. Nello specifico, è stato osservato come l’ossidazione di un residuo di metionina situato nel dominio A2 della glicoproteina impedisca il taglio proteolitico da parte di ADAMTS-13, mentre non vada ad influenzare o in alcuni casi addirittura favorisca la proteolisi di VWF da parte di proteasi leucocitarie liberate dai polimorfonucleati in seguito a stati infiammatori.

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4

Laos, Roberto, i Steven A. Benner. "Linking chemistry and biology: protein sequences". Revista de Química, 2016. http://repositorio.pucp.edu.pe/index/handle/123456789/99314.

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En los últimos veinte años el número de genomas completos que han sido secuenciados y depositados en bancos de datos ha crecido dramáticamente. Esta abundancia de información de secuencias ha servido de base para la creación de una disciplina llamada paleogenética. En este artículo, sin ahondar en algoritmos complejos, presentamos algunos conceptos clave para comprender cómo las proteínas han evolucionado con el tiempo. Luego ilustraremos como la paleogenética es utilizada en biotecnología. Estos ejemplos resaltan la conexión entre la química y la biología, dos disciplinas que quizás veinte años atrás parecían ser mucho más distintas que lo que parecen ser hoy.
In the last twenty years, the number of complete genomes that have been sequenced and deposited in data banks has grown dramatically. This abundance in sequence information has supported the creation of the discipline known as paleogenetics. In this article, without going into complex algorithms, we present some key concepts for understanding how proteins have evolved in time. We then illustrate how paleogenetic analysis can be used in biotechnology. These examples highlight the connection between chemistry and biology, two disciplines that twenty years ago seemed to be more different than what they seem to be today.

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5

Fernández, Suárez Marta. "New reporters of protein trafficking and protein-protein interactions in live cells". Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44678.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
Vita.
Includes bibliographical references.
Here, we describe our attempts to harness the exquisite specificity of natural protein and RNA enzymes to develop improved methods to study protein localization and protein-protein interactions in live cells. We first attempted to detect endogenous protein-protein interactions (PPIs) in live cells by means of a ribozyme complementation assay, but we found that the strategy was limited by the interaction affinity constraints and by low ribozyme activity in cells. We then sought to still detect interactions among endogenous proteins but in fixed cells. We devised an improved immunofluorescence (IF) technique, in which the antibodies are conjugated to an enzyme-substrate pair. We chose E. coli biotin ligase (BirA), which catalyzes the covalent ligation of biotin to a 15amino acid recognition sequence (AP). Only upon PPI would BirA be in close enough proximity to biotinylate the AP. Although the use of proximity biotinylation within the IF scheme proved challenging because of the geometric rigidity of the antibody conjugates, we later successfully applied the concept to the study of recombinant proteins in live cells, where BirA and AP were each genetically fused to the proteins of interest. We demonstrated that this method offers a combination of high spatial and temporal resolution with a low rate of false positives. We engineered the BirA/AP affinity to reduce background and eliminate false positives, while still allowing robust detection of relatively transient PPIs (half-life > 1 minute). We demonstrated that the methodology exhibits high specificity for the detection of PPIs in living mammalian cells, with a fold induction in the detected signal upon PPI of - 5-25. Using FRB-FKBP12 system as a model, the BirA/AP(-3) pair was also able to quantitatively predict interaction KIds.
(cont.) Importantly, we showed that proximity biotinylation can detect the subcellular localization of the PPI under study. We also developed a new method for site-specific labeling of proteins in live cells. Through rational design, we re-directed E. coli lipoic acid ligase (LplA) to specifically ligate an unnatural alkyl azide substrate to an engineered 22-amino acid LplA acceptor peptide (LAP) tag. The alkyl azide can then be selectively derivatized with a cyclooctyne conjugated to any probe of interest. We first demonstrated that LplA can be used to label LAP-tagged proteins with Cy3, AlexaFluor568, and biotin at the surface of living mammalian cells, and we then applied the methodology to one- and two-color cellsurface receptor labeling. Finally, we also showed that LplA can site-specifically label intracellular proteins, although the signal/background ratio still needs to be improved.
by Marta Fernández Suárez.
Ph.D.

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6

Keiser, Michael James. "Relating protein pharmacology by ligand chemistry". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378494.

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7

Ryan, C. P. "Advances in organometallic and protein chemistry". Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/20307/.

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This thesis describes two areas of scientific investigation. The first contains a description of a study on the synthesis of biotinylated and fluoresceinylated bromomaleimide based reagents. Upon synthesis, the ability of these reagents to add reversibly to cysteine containing proteins is investigated by a series of LCMS experiments. A single point mutant (L111C) of the SH2 domain of the Grb2 adaptor protein, containing a single cysteine residue, is chosen as an ideal protein for study. Thus biotinylated and fluoresceinylated mono and dibromomaleimide reagents are added to Grb2 at 0°C, 21 °C and 37 °C for 2 h at pH 7 and 8 to give protected Grb2-bromomaleimide adducts in high yield and removed using 100 eq of beta-mecaptoethanol for 4 h to return original Grb2 protein intact. Reversibility is shown to be abolished by hydrolysis of the maleimide motif, a process more prevalent in the fluoresceinylated and dibrominated maleimide reagents than for the biotinylated and monobrominated reagents. LCMS is used to investigate the insertion of these reagents reversibly into the single disulfide bridge of somatostatin, a process shown to be complete within 1 h at 21 °C and pH 6. Reduction using 100 eq of beta-mecaptoethanol is shown to take place within 1 h. Additionally no hydrolysis is observed at pH 6, suggesting that with careful control of pH, reversibility of the bromomaleimide reagents can be switched on and off. The second part of the thesis contains a study on mechanistic aspects of organopalladium catalysis, particularly the factors affecting the final reductive elimination stage of the palladium catalysed alkyl amination reaction. DFT calculations have been used to obtain a number of energy level diagrams for the potential energy surface of Pd(IBu)(neopentyl)(morpholide), the three coordinate palladium complex believed to be the species from which reductive elimination takes place. From this it has been found that two different pathways are available; reductive elimination or morpholide promoted C-H activation of the neopentyl motif, the latter of which was favoured. The reaction pathway for reductive elimination from Pd(IBu)(neopentyl)(morpholide) is compared with Pd(IBu)(phenyl)(morpholide), Pd(PCyp3)(neopentyl)(morpholide) and Pd(IBu)(methyl)(morpholide), to show that the phenyl system is considerably lower in energy. The energy requirements for (i) reductive elimination, (ii) morpholide promoted C-H activation and (iii) β-hydride elimination from Pd(IBu)(2-dimethylpropyl)(morpholide) are compared, and shown to be in the order of energy (i)>(ii)>(iii). Additionally, the rate of reaction increases as the number of available reaction sites increases, again making reductive elimination the least favoured process. Offline ESI(+)-MS analysis have been used to monitor the reaction progress of Pd(IBu)(neopentyl)(morpholide) and Pd(IBu)(phenyl)(morpholide) three coordinate complexes. Whilst transient palladium-bound species can be observed for Pd(IBu)(phenyl)(morpholide), for Pd(IBu)(neopentyl)(morpholide) this is not the case, an artefact of the superiority of the sp2 phenyl system over the sp3 alkyl system.

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8

Liu, Xingquan 1959. "Import of proteins into mitochondria : biogenesis of the uncoupling protein and identification of a mitochondrial signal peptide binding protein". Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74310.

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The inner membrane uncoupling protein (UCP) of rat brown fat mitochondria has been imported into rat heart mitochondria in vitro. Two import signals have been detected in UCP. The intrinsic membrane insertion information of UCP has been abrogated by a signal sequence fused in front of UCP, resulting in the rerouting of UCP into the matrix. Following removal of the signal sequence from the hybrid protein, the UCP moiety remained in the soluble matrix space indicating an incompatibility of UCP insertion into the inner membrane from the matrix side.
An integral mitochondrial membrane protein (p30) that binds a mitochondrial signal peptide in intact mitochondria in vitro has been purified by an affinity approach. The protein has been identified as a member of the ADP/ATP carrier (AAC) family based on both immunoblotting and peptide mapping. The irreversible association of the signal peptide with AAC in intact mitochondria has been correlated with inhibition of protein import into the organelle.

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9

Ju, Yue. "MASS SPECTROMETRIC STUDY OF PROTEIN AND PROTEIN LIGAND COMPLEXES". The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449219266.

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10

Bennett, Matthew Stuart. "Crystallography of biomolecular complexes, revealing protein-nucleoside, protein-protein acid-drug interactions". Thesis, King's College London (University of London), 2002. https://kclpure.kcl.ac.uk/portal/en/theses/crystallography-of-biomolecular-complexes-revealing-proteinnucleoside-proteinprotein-aciddrug-interactions(4a85b5cd-8a9a-4969-bee3-95fbe46e4257).html.

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11

Almonacid, Coronado Daniel Eduardo. "The chemistry and evolution of protein catalysis". Thesis, University of Cambridge, 2008. https://www.repository.cam.ac.uk/handle/1810/252074.

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12

Matsuzaki, Hideki. "Protein and Enzyme Chemistry of Cathepsin A". Kyoto University, 1998. http://hdl.handle.net/2433/182423.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7562号
農博第1023号
新制||農||772(附属図書館)
学位論文||H10||N3212(農学部図書室)
UT51-99-A248
京都大学大学院農学研究科農芸化学専攻
(主査)教授林力丸, 教授天知輝夫, 教授清水昌
学位規則第4条第1項該当

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13

Lauffer, Benjamin E. L. "Protein interactions mediating endocytic recycling of G protein-coupled receptors". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3339215.

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14

Bayro, Marvin J. "Protein MAS NMR methodology and structural analysis of protein assemblies". Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57800.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Methodological developments and applications of solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, with particular emphasis on the analysis of protein structure, are described in this thesis. MAS NMR studies of biomolecules ranging from model peptides and proteins in crystalline form to amyloid fibrils and whole bacterial organelles are reported. The methods presented include novel pulse sequences and optimized pulse sequence elements, experimental approaches designed for multiple-spin systems, a protocol for efficient sequential resonance assignment of proteins in the solid state, and techniques to determine the inter-molecular organization of amyloid fibrils formed by moderately sized proteins. Notably, an efficient dipolar recoupling technique, bandselective radio frequency-driven recoupling (BASE RFDR), is introduced and combined with alternating 13C-12C labeling to yield highly sensitive 13C-13C correlation spectra between distant nuclei in proteins. Various applications of the BASE RFDR scheme are presented, including protein resonance assignment, determination of tertiary structure of amyloid fibrils, and variable-temperature studies of protein dynamics. The main biological systems analyzed are amyloid fibrils formed by the SH3 domain of P13 kinase (P13-SH3) and intact gas vesicles from anabaena flos-aquae, for which atomic-level structural information was previously unavailable. P13-SH3 (86 residues) is a system thoroughly studied as a model of protein misfolding and amyloid formation by a natively globular protein. Gas vesicles are bacterial buoyancy organelles, with walls composed almost entirely by a single protein (GvpA, 70 residues), whose formation and structure constitute a highly intriguing biophysical problem. Nearly complete 13C and 'IN resonance assignments and the molecular conformations of the polypeptide backbones of both P13-SH3 and GvpA have been obtained via MAS NMR spectroscopy, enabling the proposal of models for the structure of these two protein assembly systems. In addition, the tertiary structure of P13-SH3 amyloid fibrils has been elucidated by the application of novel methodology introduced in this thesis. Finally, investigations regarding the effects of temperature and protein dynamics on MAS NMR experiments and biomolecular dynamic nuclear polarization studies are presented.
by Marvin J. Bayro.
Ph.D.

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Measey, Thomas J. Schweitzer-Stenner Reinhard. "Unfolded, misfolded, and self-organized short alanine-rich peptides : implications for fundamental science, human disease, and biotechnology /". Philadelphia, Pa. : Drexel University, 2010. http://hdl.handle.net/1860/3317.

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Lian, Wenlong. "DEVELOPMENT AND OPTIMIZATION OF PROTEIN-PROTEIN INTERACTION INHIBITORS BY COMBINATORIAL AND MEDICINAL CHEMISTRY". The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449161031.

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Spatara, Michelle L. "Protein folding and aggregation in vitro and in vivo". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 162 p, 2009. http://proquest.umi.com/pqdweb?did=1892027491&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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Lee, Joongoo. "A semisynthetic protein nanoreactor for single-molecule chemistry". Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:b0c61278-5483-44b7-a662-f079c0f2c23f.

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The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.

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19

Apgar, James R. (James Reasoner). "Modeling the flexibility of alpha helices in protein interfaces : structure based design and prediction of helix-mediated protein-protein interactions". Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/43778.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
Vita.
Includes bibliographical references.
Protein-protein interactions play an essential role in many biological functions. Prediction and design of these interactions using computational methods requires models that can be used to efficiently sample structural variation. This thesis identifies methods that can be used to sample an important sub-space of protein structure: alpha helices that participate in protein interfaces. Helices, the global structural properties of which can be described with only a few variables, are particularly well suited for efficient sampling. Two methods for sampling helical backbones are presented: Crick parameterization for coiled coils and normal-mode analysis for all helices. These are shown to capture most of the variation seen in the PDB. In addition, these methods are applied to problems in protein structure prediction and design. Normal-mode analysis is used to design novel nanomolar peptide inhibitors of the apoptosis-related Bcl-2 family member, Bcl-xL, and a modification of Crick Parameterization is used to predict the binding orientation of dimeric coiled coils with greater than 80% accuracy. Finally, this study addresses the increase in computational time required by flexible-backbone methods and the use of cluster expansion to quickly map structural energies to sequence-based functions for increased efficiency.
by James R. Apgar.
Ph.D.

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20

Si, Chao. "Theoretical Study of Intermolecular Interactions in Protein-Drug Binding and Protein Folding". University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1341632548.

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Shih, Yu-Hsuan. "Protein Disaggregation and Degradation by Clp ATPase Nanomachines during Protein Quality Control". University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1512045252522582.

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Phillips, Angela Marie Ph D. Massachusetts Institute of Technology. "Chaperoning viral protein evolution". Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/118276.

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Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Preventing viral pandemics and developing effective antiviral therapeutics demands understanding the molecular mechanisms that both potentiate and constrain viral evolution. The rapid evolution of viruses is mediated in part by their high mutation rates, enabling resistance to antiviral drugs, seasonal vaccines, and innate and adaptive immune responses. Fortunately for us, the same mutations responsible for resistance are often biophysically deleterious to viral proteins. Thus, viral evolution is inherently constrained by the proper folding of viral proteins into functional, stable conformations. In cells, protein folding and homeostasis are assisted by complex networks of chaperones and quality control machinery. Though the evolutionary implications of most chaperones and quality control factors remain unexplored, the HSP90 chaperone can buffer and potentiate the phenotypic effects of mutations in endogenous client proteins in bacteria, fungi, plants, and other eukaryotic organisms. Viruses acquire mutations at a rate several orders of magnitude above that of the aforementioned organisms, yet they do not encode any machinery to assist destabilized protein variants to their folded, functional conformations. However, viral proteins are known to interact with host chaperones and quality control machinery. My graduate work has focused on determining whether and how host proteostasis machinery modulates viral protein evolution. First, I employed a serial passaging approach to evolve influenza in host cells with remodeled proteostasis capacities, revealing that cytosolic host proteostasis capacity is indeed a critical determinant of influenza evolutionary trajectories. This work motivated systematic quantification of influenza protein mutational tolerance upon perturbation of host proteostasis, for which I applied deep mutational scanning to comprehensively profile the mutational tolerance of influenza nucleoprotein and hemagglutinin in modulated cytosolic and endoplasmic reticulum (ER) folding environments, respectively. The nucleoprotein work provides the first experimental evidence that host chaperones can enhance the accessibility of biophysically deleterious, adaptive viral protein variants. The hemagglutinin work establishes evolutionary implications for the ER proteostasis machinery, and demonstrates that ER proteostasis mechanisms enhance mutational tolerance across the entire HA protein. Overall, it is clear that host chaperones and quality control machinery crucially impact viral protein fitness, and likely also impact the fitness of endogenous variants.
by Angela Marie Phillips.
Ph. D. in Biological Chemistry

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23

GULOTTA, Maria Rita. "Computational methodologies applied to Protein-Protein Interactions for molecular insights in Medicinal Chemistry". Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/479127.

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In living systems, proteins usually team up into “molecular machinery” implementing several protein-to-protein physical contacts – or protein-protein interactions (PPIs) – to exert biological effects at both cellular and systems levels. Deregulations of protein-protein contacts have been associated with a huge number of diseases in a wide range of medical areas, such as oncology, cancer immunotherapy, infectious diseases, neurological disorders, heart failure, inflammation and oxidative stress. PPIs are very complex and usually characterised by specific shape, size and complementarity. The protein interfaces are generally large, broad and shallow, and frequently protein-protein contacts are established between non-continuous epitopes, that conversely are dislocated across the protein interfaces. For this reason, in the past two decades, PPIs were thought to be “undruggable” targets by the scientific research community with scarce or no chance of success. However, in recent years the Medicinal Chemistry frontiers have been changing and PPIs have gained popularity amongst the research groups due to their key roles in such a huge number of diseases. Until recently, PPIs were determined by experimental evidence through techniques specifically developed to target a small group of interactions. However, these methods present several limitations in terms of high costs and labour- and time-wasting. Nowadays, a large number of computational methods have been successfully applied to evaluate, validate, and deeply analyse the experimentally determined protein interactomes. In this context, a high number of computational tools and techniques have been developed, such as methods designed to construct interaction databases, quantum mechanics and molecular mechanics (QM/MM) to study the electronic properties, simulate chemical reactions, and calculate spectra, and all-atom molecular dynamics simulations to simulate temporal and spatial scales of inter- and intramolecular interactions. These techniques have allowed to explore PPI networks and predict the related functional features. In this PhD work, an extensive use of computational techniques has been reported as valuable tool to explore protein-protein interfaces, identify their hot spot residues, select small molecules and design peptides with the aim of inhibiting six different studied PPIs. Indeed, in this thesis, a success story of in silico approaches to PPI study has been described, where MD simulations, docking and pharmacophore screenings led to the identification of a set of PPI modulators. Among these, two molecules, RIM430 and RIM442, registered good inhibitory activity with IC50 values even within the nanomolar range against the interaction between MUC1 and CIN85 proteins in cancer disease. Furthermore, computational alanine scanning, all-atom molecular dynamics simulations, docking and pharmacophore screening were exploited to (1) rationally predict three potential interaction models of NLRP3PYD-ASCPYD complex involved in inflammatory and autoimmune diseases; (2) identify a potentially druggable region on the surface of SARS-CoV-2 Spike protein interface and select putative inhibitors of the interaction between Spike protein and the host ACE2 receptor against COVID-19 (CoronaVIrus Disease 2019); (3) investigate intramolecular modifications as a consequence of a point mutation on C3b protein (R102G) associated with the age-related macular degeneration (AMD) disease; (4) design non-standard peptides to inhibit transcriptional events associated with HOX-PBX complex involved in cancer diseases; and (5) to optimise a patented peptide sequence by designing helix-shaped peptides embedded with the hydrogen bond surrogate approach to tackle cocaine abuse relapses associated with Ras-RasGRF1 interaction. Although all the herein exploited techniques are based on predictive calculations and need experimental evidence to confirm the findings, the results and molecular insights retrieved and collected show the potential of the computer-aided drug design applied to the Medicinal Chemistry, guaranteeing labour- and time-saving to the research groups. On the other hand, computing ability, improved algorithms and fast-growing data sets are rapidly fostering advances in multiscale molecular modelling, providing a powerful emerging paradigm for drug discovery. It means that more and more research efforts will be done to invest in novel and more precise computational techniques and fine-tune the currently employed methodologies.

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Angell, Yu Li. "Triazole based peptidomimetics for mimicking protein-protein hot spots". [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1432.

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Long, Jiafu. "Supramodular nature of neuronal scaffolding proteins /". View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20LONG.

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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004.
Includes bibliographical references (leaves 167-184). Also available in electronic version. Access restricted to campus users.

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Ryan, Jeremy Adam. "Design and synthesis of probes for detection of protein-protein interaction and RNA localization". Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33657.

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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.
Includes bibliographical references.
The use of the ketone biotin - benzophenone-biotin hydrazide system for detecting the formation of cyan fluorescent protein and NF-kappaB p50 dimers was assessed. A series of benzophenone-based probes were synthesized and tested for photocrosslinking activity to investigate the efficiency of photocrosslinking in these systems. Three series of small molecule probes were synthesized for the selection of ribozymes from a random sequence pool. Solid-phase immobilized fluorescein and fluorescein phosphates were synthesized for the indirect selection of a fluorescein phosphatase ribozyme. A corresponding thiophosphate analog was created for the in-gel selection of a thiophosphatase ribozyme via APM-PAGE. Finally, a series of fluorescein-nucleoside phosphate conjugates was designed and synthesized for use in the solution phase preparation of a fluorogenic ribozyme substrate, and later immobilization of this substrate on a silyl resin for direct ribozyme selection.
by Jeremy Adam Ryan.
S.M.

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27

Sarkar, Mohosin M. "Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility". The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.

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Füllen, Georg Karl-Heinz. "Protein engineering and pattern recognition". Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/17354.

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Wavreille, Anne-Sophie Marie. "SRC homology 2 domain proteins binding specificity from combinatorial chemistry to cell-permeable inhibitors /". Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1164738844.

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Cheng, Yu-Chia. "Effects of salts on the phase behavior of proteins and protein mixtures". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 281 p, 2008. http://proquest.umi.com/pqdweb?did=1456290961&sid=10&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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Reyes, Samuel Onofre J. "Expanding beta-turn analogs for mimicking protein-protein hot spots". Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1748.

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Brophy, Megan Brunjes. "Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin". Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/98823.

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Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
The human innate immune system responds to bacterial and fungal pathogens by releasing the metal-chelating protein calprotectin (CP) at sites of infection and in the upper layers of the epidermis. CP is a Mn(II)- and Zn(ll)-binding protein. The work described in this thesis elucidates the metal-binding properties of CP, and correlates these properties with in vitro growth inhibition of bacteria and fungi. We report that the metal-binding properties of CP are modulated by Ca(ll), and we propose a working model in which CP responds to physiological Ca(Il)-ion gradients to become a potent Zn(ll)- and Mn(Il)-chelating agent in the extracellular space. Individual chapter summaries follow. Chapter 1: Bioinorganic Chemistry of the Host Pathogen Interaction. Transition metal ions are required for all forms of life. During the course of infection, pathogenic microorganisms must acquire transition metals from the host. Three metals of interest from this standpoint are iron, zinc, and manganese. This chapter describes bacterial metal-ion homeostasis machineries, and metal-requiring processes with a focus on Zn(II) and Mn(II). This chapter then highlights the S100 family of Ca(ll)-binding proteins and discuses the Zn(Il)-, Cu(ll)-, and Mn(Il)-binding properties of S100B, S100A12, S100A7, S10OA15, and S100A8/S100A9. Finally, an overview of the scope of this thesis is presented. Chapter 2: Calcium Ion Gradients Modulate the Zinc(Il) Affinity and Antibacterial Activity of Human Calprotectin. Calprotectin (CP) is a human neutrophil protein that is produced and released by neutrophils at sites of infection, where it prevents the growth of microorganisms by sequestering bioavailable zinc(II) and manganese(II). In this chapter, we present metalbinding studies to elucidate the Zn(ll)-binding properties of CP. We report unique optical absorption and EPR spectroscopic signatures for the interfacial His 3Asp and His 4 sites of human CP by using Co(II) as a spectroscopic probe. Zinc competition titrations employing colorimetric and fluorimetric Zn(II) sensors establish that CP coordinates two Zn(II) ions / CP heterodimer. The Ca(ll)-insensitive Zn(ll) sensor ZP4 is used to determine the Kd of CP for Zn(II) in Ca(Il)-deplete and Ca(Il)-replete conditions. These competition titrations afford apparent Kdsitel = 133 58 pM and Kdsite2 = 185 219 nM in the absence of Ca(II). In the presence of excess Ca(Il) these values decrease to Kd,sitel 5 10 pM and Kd,site2 : 240 pM. In vitro antibacterial assays indicate that the metal-binding sites and Ca(ll)-replete conditions are required to inhibit the growth of Gram-negative and Gram-positive bacteria. We propose a model in which Ca(II) ion gradients modulate the antibacterial activity and Zn(Il)-binding properties of human CP. Chapter 3: High-Affinity Manganese Coordination by Human Calprotectin Is Calcium- Dependent and Requires the Histidine-Rich Site at the Dimer Interface. In this chapter, we report that the His 4 motif at the S10OA8/S100A9 dimer interface of CP is required for high-affinity Mn(II) coordination. We identify a low-temperature EPR spectroscopic signal for this site that is consistent with high-spin Mn(II) in an octahedral coordination sphere. This site could be simulated with zero-field splitting parameters D = 270 MHz and EID = 0.30 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that (A8)Hisl7, (A8)His27, (A9)His9l, (A9)His95 and two as-yet unidentified ligands coordinate Mn(ll) at site 2. These studies support a model in which CP responds to Ca(ll) ion gradients to become a potent metal-ion chelator in the extracellular space. Chapter 4: Contributions of the C-terminal Tail of S100A9 to High-Affinity Manganese Binding by Human Calprotectin. This chapter examines the role of the S100A9 C-terminal tail to high-affinity Mn(ll) coordination by human CP. We present a 16-member mutant family with mutations in the S100A9 C-terminal tail (residues 96-114), which houses three histidine and four acidic residues, to evaluate its contribution to Mn(ll) sequestration. These studies confirm that two His residues at positions 103 and 105 complete the octahedral coordination sphere of CP in solution. Appendix 1: Sequence Alignments of Transition-Metal Binding S100 Proteins. Sequence alignments of S100A7, S100A8, S100A9, S100A12, S100A15, and S100B proteins from multiple organisms are presented. Appendix 2: Characterization of CP Mutant Proteins by Circular Dichroism and Analytical Size Exclusion Chromatography. Additional characterization of CP and mutant proteins employed in Chapters 2-4 is presented. Appendix 3: Structures of Sensors Used In this Work. The structures of Zincon, MagFura-2, Zinpyr-1, and Zinpyr-4 are presented. Appendix 4: Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium. This appendix represents a collaborative work with the Drennan Lab (MIT) and Britt Lab (UC Davis) to study the Mn(Il)-CP complex in low- and high-Ca(II) conditions. We report a crystal structure of Mn(Il)-, Ca(Il)-, and Na(l)-bound CP with Mn(II) exclusively coordinated to the His6 motif. Electron spin-echo envelope modulation and electron-nuclear double resonance experiments demonstrate that the six coordinating histidine residues are spectroscopically equivalent. The observed 15N ( = %/h)y perfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating E-nitrogen of the imidazole ring of each histidine (A = [3.45, 3.71, 5.91] MHz) and the distal 6-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the affinity of CP for Mn(II) drops by two to three orders of magnitude, and Mn(II) coordinates to the His6 site as well as other sites on the protein.
by Megan Brunjes Brophy.
Ph. D. in Biological Chemistry

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33

Higgs, C. "A computational study of the G-protein-G-protein coupled receptor interaction". Thesis, University of Essex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324216.

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Lee, John Edwin 1965. "Molecular orientation distributions in adsorbed protein films". Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282448.

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Understanding and controlling protein adsorption is fundamentally important to the successful development of synthetic biomaterials and implantable chemical sensing devices. However, the study of protein adsorption, and in particular, orientation in protein thin films has been hampered by inadequate methods with which to study weakly absorbing thin protein films. In this work, Integrated Optical Waveguide-Attenuated Total Reflection Linear Dichroism (IOW-ATR LD) coupled with fluorescence emission anisotropy was used to study: (1) the orientation of Mb as a function of the adsorbent surface and surface coverage, and (2) the orientation distributions of cyt c adsorbed to various surfaces. It was found that the average molecular orientation of an adsorbed protein film: (1) is dependent on the chemical and/or physical characteristics of the adsorbent surface, and (2) is dependent on the protein surface coverage. It was also determined that the macroscopic order of an adsorbed protein film is dependent on the number of different types of protein-surface interactions in a given system. If there is one dominant type of interaction between the between the protein and the surface which is limited to a specific region on the surface of the protein, an ordered protein film will be produced. However, as the number of the type of protein-surface interactions increases, the distribution of orientations also increases, leading to a disordered film. Finally, a broadband waveguide coupling device has been developed which allows spectroscopic measurements of submonolayer to monolayer surface coverages of proteins to be performed.

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35

Lopac, Senja Katarina. "Polymer chemistry effects on protein release and immune activation". [Ames, Iowa : Iowa State University], 2008.

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Morimoto, Koichi. "Studies on Highly Sensitive Enzyme Immunoassays by Protein Chemistry". Kyoto University, 1997. http://hdl.handle.net/2433/157104.

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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第9633号
論農博第2147号
新制||農||747(附属図書館)
学位論文||H9||N3072(農学部図書室)
UT51-97-L210
(主査)教授井上國世, 教授松野隆一, 教授廣瀬正明
学位規則第4条第2項該当

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37

Whelan, Helen A. "Calmodulin Dependent Protein Kinase II - A Sulfhydryl Chemistry Study /". The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu148793151262033.

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Dowman, Luke James. "Expanding the Scope of Peptide and Protein Modification Chemistry". Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23402.

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Peptides and proteins are nature’s functional biomolecules which orchestrate virtually all the biological processes that define life. Posttranslational modifications (PTMs) of the side chains of amino acids within peptides and proteins represent a means to expand the repertoire of protein structure and function from an otherwise concise genome. The role of these modifications is increasingly recognised as critical for the structure and function of proteins and underpins the burgeoning protein therapeutics (“biologics”) market. Access to these molecules in homogeneous form, however, has remained challenging. Specifically, the generation of inseparable mixtures of heterogeneously modified proteins in mammalian or insect expression systems, and issues regarding the regioselectivity of bioconjugation chemistries have plagued access to these valuable biomolecules. This thesis will describe novel chemical methods for accessing such modified peptide and protein targets in homogeneous form. Specifically, Chapter 2 will describe the synthesis of suitably protected analogues of the modified amino acid, sulfotyrosine, and their incorporation into known sulfated anti-thrombotic peptides for the purpose of increasing acid-stability while retaining biological activity. Chapters 3 and 4 describe the serendipitous discovery and subsequent development of a novel photocatalytic reaction manifold for the contraction of symmetric or asymmetric peptide and protein diselenides to their corresponding selenoethers with formal extrusion of a Se atom. Preliminary exploration of the mechanism of this transformation is discussed alongside the application of the reaction to both the dimerisation and modification of selenopeptides and selenoproteins. Several selenoprotein targets for modification under this manifold are then chosen for preliminary studies into their recombinant production through an evolved selenocysteine-dependent cell line operating under a nonsense codon suppression system.

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Bietlot, Henri P. "Characterization of the insecticidal protein from Bacillus thuringiensis: The importance of DNA-protein interactions". Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6598.

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Many strains of Bacillus thuringiensis produce a crystalline inclusion during sporulation which is toxic to insect larvae. The major component of crystals toxic to lepidopteran larvae is a 130-kDa protein, the protoxin. Following ingestion by susceptible insect larvae, protoxin is proteolysed by larval gut proteinases to yield a 58-70 kDa toxic fragment, toxin. A procedure was developed to prepare purified toxin for chemical characterization. Toxin generated by bovine trypsin was shown to be composed of the amino acid residues that span position 29-623 of the protoxin. The results obtained from competitive labelling experiments on the protoxin show that the functional groups of the lysine and tyrosine residues do not exhibit regular titration behaviour over the pH range of 7 to 10. These results indicate that the majority of these groups are not free in solution but are involved in inter and intra molecular interactions. During purification by ion exchange chromatography of the bovine generated toxin, it was discovered that the toxin could be separated into two components. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contain 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of greater than $2\times 10\sp6$ Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the carboxyl terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.

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Brenner, Rachel Natanya. "Structure/function studies of protein phosphatases". Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/37550.

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Seneviratne, Herana Kamal. "Towards Understanding Dirigent Protein Function". Thesis, Washington State University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10269677.

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Plants produce a dazzling collection of chemical compounds with great structural and functional diversity that are known as specialized metabolites which have important roles in plant defense responses. Most significantly, these chemical entities serve as rich and important source of biologically active pharmaceuticals. Although much has been known about the immense importance of these specialized metabolites, little is known about how they are produced in plants.

The evolution of the biochemistry of vascular plants depend extensively upon unique phenoxy radical-radical coupling reactions to produce different chemical compounds. In this regard, a group of proteins called dirigent proteins (DPs) control the stereoselectivities of above-mentioned coupling processes to engender a class of specialized metabolites known as lignans. In particular, lignans represent a large class of plant specialized metabolites which exhibit a broad range of physiological functions and potential medicinally important therapeutic properties. Even though there are many dirigent homologs in the plant kingdom, we do not know the precise functions of these homologous proteins.

This dissertation describes the elucidation of the biochemical function of a dirigent homolog, i.e. PsDRR206 and localization of phytoalexin pathway in pea (Pisum sativum) pods using high-resolution mass spectrometry techniques. From the studies described herein, the metabolite associated with PsDRR206 gene was identified as pinoresinol monoglucoside. Additionally, the localization of selected phytoalexins including pinoresinol monoglucoside as well as (+)-pisatin was determined as the endocarp epidermal cell layer of pea pod tissue. The results from these studies provide new insights for plant biochemistry research through gaining the knowledge of important biological processes occurring in nature.

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Pham, Thi Minh Hai. "Protein extraction using reverse micelles". Thesis, University of Greenwich, 2015. http://gala.gre.ac.uk/18122/.

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Reverse micelles are self-organized aggregates formed by a surfactant in a non-polar solvent or oil. The presence of a water pool in the polar core of reverse micelles is of considerable advantage in protein extraction. A lot of researches have been done with ionic reverse micelles applied in protein extraction. However, this ability of non-ionic reverse micelles has not been fully understood and therefore requires more research. In this project, different surfactants (anionic AOT, cationic CTAB, non-ionic triblock L61 copolymer) were investigated for their ability to form RM and for their application in protein extraction. It was found that lysozyme could be extracted using an AOT RMS, but not with a CTAB RMS. For the first time, an aromatic solvent, p-xylene, was used for the extraction of lysozyme and it was found that the AOT in p-xylene RM system resulted in the higher lysozyme activity (73.81 %) compared to an AOT/isooctane RM system (43.2 %). The effect of different salts (KCl, KF, KBr) on the FE and BE of BSA was investigated using the CTAB in mixture of 1-bromooctane, 1-hexanol and petroleum ether. The results indicated that KCl gave the highest extraction efficiency of 64 % as compared to around 40 % with both cases of KF and KBr. The secondary structure of extracted BSA was maintained with KCl only. L61 pluronics polymers was investigated for its reverse micelles forming ability and it was established that small reverse micelles with a maximum W0 of 4 was formed. Because of the small size of L61 reverse micelles, lysozyme could not be extracted but was precipitated out when combined with the co-surfactant AOT. The activity of the recovered lysozyme from the precipitate was maintained (66% as compared with native lysozyme). Moreover, if L61 was used as a co-surfactant with AOT reverse micelles, extraction efficiency was improved (88 %) and the activity of the extracted ly-sozyme was increased (56 %) as compare to extraction with an AOT system alone (46 %). These studies thus gives useful insights in the role of individual and mixed surfactant systems in the extraction and precipitation of proteins.

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Schmitt, Jaclyn L. "Understanding timing| Conservation between the circadian protein period and the C. elegans developmental timing protein lin-42". Thesis, University of California, Santa Cruz, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1551325.

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Timing of development, metabolic regulation, and longevity are crucial elements in optimizing physiological functions to, and within, our environment. The synchronization of our internal clocks to the twenty-four hour day of our environment aids in anticipatory and protective measures on the molecular level. Dysregulation of this internal clock, known as the circadian clock, has been linked to various cancers, diabetes and heart failure. Mental ailments such as alcoholism and bipolarism can be magnified through dysregulation of our circadian rhythms. The output of circadian time keeping is still being explored, including the link to longevity. To further our understanding of clock functions through molecular structure, comparisons between biological time keeping methods are vital. On the molecular level of the circadian clock, one of the core negative feedback loop proteins is PERIOD. The complex timing of PERIOD transcription and protein accumulation directly contributes to setting the circadian clock. Within PERIOD protein, the functions of the homo- and heterodimerizing PERIOD-ARNT-SIM (PAS) domain to facilitate nuclear localization, and possibly many of the PERIOD output functions, are still being understood. Another protein that contains this canonical PAS domain is the nematode C. elegans development timing protein LIN-42. Although C. elegans are not known to have circadian rhythms, LIN-42 shares many motif and functional similarities to PERIOD. The development of C. elegans larva is repressively regulated, or gated, by LIN-42. Additionally, LIN-42 regulates entry into quiescent states during larval devolvement when environmental conditions are stressful. Considering the functions of LIN-42 within development of specialized stem cells, known as seam cells, and the recent discovery of the functions of PERIOD within the development of our own stem cells; a molecular comparison of LIN-42 and PERIOD will facilitate our understanding of the associated output functions of these proteins. Specifically the N-terminal regions of PER and LIN-42 share well-folded structural domains, and are the focus of this thesis. The forms of PERIOD and LIN-42 that share the most sequence and functional homology are PER2 and LIN-42b. Direct comparison of the similarities and differences between these two proteins on the molecular level will shed light on biological time keeping.

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Watkins, Andrew M. "An in silico pipeline for the design of peptidomimetic protein-protein interaction inhibitors". Thesis, New York University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10188557.

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Protein-protein interactions have historically been branded “undruggable” due to their intrinsic challenges above and beyond protein-small molecule interactions. Incrementally, system after system has been approached by a variety of specialized design strategies. Still, the vast majority of interactions are intractable, and the profusion of individualized strategies leave few general approaches that might be able to extend to recalcitrant systems.

The ecosystem of tools available for developing inhibitors of protein-protein interactions suggests a potential modular strategy for proceeding from protein structure to plausible interaction inhibitors. My dissertation describes an analysis of all the protein-protein interactions containing key interfacial structural motifs found in protein structures catalogued by the Protein Data Bank. This work provides both data on extant protein interactions and specific conclusions regarding directions for further peptidomimetic design. We describe the incorporation of our lab’s peptidomimetic scaffolds into Rosetta and the validation of those methods against valuable biological systems. Finally, I chronicle substantial extension to Rosetta’s capacity to accurately model and design peptidomimetic structures.

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Male, Abigail. "Identification of inhibitors of protein-protein interactions essential for virulence in pathogenic bacteria". Thesis, University of Southampton, 2014. https://eprints.soton.ac.uk/369351/.

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There is a continous requirement for broad-spectrum post-exposure antibiotic therapeutics. Meeting this challenge relies on the production of compounds that successfully disrupt bacterial systems identified as both conserved and essential. Here, inhibitors of protein-protein interactions involved in the Phage shock protein response and toxin internalisation, within Burkholderia pseudomallei and Bacillus anthracis, respectively have been identified. This was achieved using a high-throughput screen that combines a bacterial reverse two-hybrid system and an intein-mediated method for the generation of cyclic peptide libraries. A reverse two-hybrid system for Burkholderia pseudomallei PspA homodimerisation was constructed, alongside a heterodimeric system for the interaction between Bacillus anthracis protective antigen and the mammalian receptor, capillary morphogenesis gene-2. From both systems a series of peptide sequences were identified with potential inhibitory activity within the reverse two-hybrid system. These compounds were synthesised and their activity assessed using a selection of in vitro assays. This study identified two cyclic peptides sequences active in the reverse two-hybrid system against PspA oligomerisation; which were not active in vitro. In contrast, three linear peptides were isolated that demonstrated the ability to disrupt the interaction between protective antigen and the mammalian receptor, with one binding specifically to the receptor. This linear inhibitor provides the foundation for the development of a more potent antimicrobial for the arsenal against Bacillus anthracis.

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Luechapanichkul, Rinrada. "Determination of the Sequence Specificity and Protein Substrates of Protein Phosphatases". The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398868380.

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Friedli, Georges-Louis. "Interaction of deamidated soluble wheat protein (SWP) With other food proteins and metals". Thesis, University of Surrey, 1996. http://epubs.surrey.ac.uk/2204/.

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Stiving, Alyssa Quencer. "Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure". The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1587409993097519.

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Cooper, Jahan. "Optimizing the Potency of a Bicyclic Peptide Inhibitor of the Ras-Raf Protein-Protein Interaction via Combinatorial Screening". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu152570020664904.

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Mealman, Tiffany Diane. "Metal Transfer And Protein-Protein Interactions In The CusCFBA CU(I)/AG(I) Efflux System Of E. Coli". Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/312754.

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Copper homeostasis in Escherichia coli is mediated in part by a copper sensing system (the Cus system) that includes a tripartite protein complex, CusCBA, which spans both the inner and outer membranes of E. coli. The inner membrane protein, CusA, belongs to the RND superfamily of protein transporters and couples the export of copper ions with an influx of protons. CusB, the membrane fusion protein, is a soluble protein that forms a complex with CusA and is believed to interact with CusC, the outer membrane factor protein. CusF, the fourth component of this system, is a small periplasmic metallochaperone that delivers metal to the CusCBA pump. The research presented in this work describes the elucidation of protein-protein interactions and metal transfer between components of the Cus system. Chemical cross-linking and mass spectrometry reveal that CusF and CusB interact at their metal-binding sites. The N-terminal region of CusB, which includes the metal-binding site, is characterized utilizing a variety of biochemical and molecular tools and demonstrates metal-binding and metal transfer with CusF. Cross-linking experiments and XAS preliminary results reveal a novel interaction between CusF and CusA, which supports our theory that CusA acquires metal from CusF rather than CusB. Together, these findings aid in our understanding of the mechanism of metal transport and the molecular details involved in protein-protein interactions within CusCFBA.

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