Tesis: "Intrinsically disordered"

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Wong, Eric Tsz Chung. "Electrostatics in intrinsically disordered proteins". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43451.

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2

Crabtree, Michael David. "Probing order within intrinsically disordered proteins". Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/270303.

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Decades have passed since the realisation that a protein’s amino acid sequence can contain all the information required to form a complex three-dimensional fold. Until recently, these encoded structures were thought to be crucial determinants of protein function. Much effort was directed to fully understand the mechanisms behind how and why proteins fold, with natively unfolded proteins thought to be experimental artefacts. Today, the field of natively unfolded – or so-called intrinsically disordered – proteins, is rapidly developing. Protein disorder content has been positively correlated with organismal complexity, with over thirty percent of eukaryotic proteins predicted to contain disordered regions. However, the biophysical consequences of disorder are yet to be fully determined. With the aim of addressing some of the outstanding questions, the work described in this thesis focuses on the relevance of structure within disordered proteins. Whilst populating a variety of conformations in isolation, a subset of disordered proteins can fold upon binding to a partner macromolecule. This folded state may be present within the ensemble of conformations sampled by the unbound protein, opening the question of what comes first: folding or binding? Protein engineering techniques were employed to alter the level of residual ‘bound-like’ structure within the free conformational ensemble, and the consequences on coupled folding and binding reactions were investigated. Resultant changes in the rate of association are easily imaginable; yet, this work demonstrates that the majority of the observed changes in binding affinity were due to alterations in the rate of dissociation, thus altering the lifetime of the bound complex. Promiscuous binding is a touted advantage of being disordered. If many disordered proteins, each with their own conformational ensemble, can bind and fold to the same partner, then where is the folding component encoded? Does the partner protein template the folding reaction? Or, is the folding information contained within the disordered protein sequence? Utilising phi-value analysis on the BCL-2 family of proteins, residues in the disordered sequence were probed to ascertain which form contacts at the transition state of the reaction. Comparison with phi-value analyses of alternative pairs – sharing either the ordered or disordered protein – provides insight into the encoding of these interactions. In the context of a bimolecular reaction, the amino acid sequence of the disordered protein was shown to determine the interactions within the transition state. Thus, analogous to the discovery from decades’ past, it is the sequence of the protein that folds which encodes its pathway, even when binding is a prerequisite.

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3

Zhang, Yizhi. "Intrinsically disordered proteins in Chlamydomonas reinhardtii". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0290/document.

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Les objectifs de cette thèse étaient d'apporter une percée conceptuelle pour une compréhension en profondeur des mécanismes moléculaires des protéines intrinsèquement désordonnées (IDPs) et de leurs rôles dans la physiologie cellulaire de Chlamydomonas reinhardtii. La combinaison d’approches expérimentale et bioinformatique m’a permis d’identifier 682 protéines thermorésistantes chez C. reinhardtii. Parmi celles-ci, 299 protéines sont systématiquement prédites comme potentielles IDP par quatre algorithmes de prédiction de désordre. Nos résultats indiquent que le pourcentage désordonné moyen de ces protéines prédites comme étant des IDPs est d'environ 20%, et la plupart d'entre elles (~70%) sont adressées à d'autres compartiments que la mitochondrie et le chloroplaste. Leur composition en acides aminés est biaisée par rapport à d'autres IDPs de la base de données de protéines désordonnées (DisProt). Ces IDPs potentielles jouent des fonctions moléculaires diverses, et 54% d'entre elles sont des cibles de phosphorylation.Notre travail a également augmenté l’état des connaissances sur l'adénylate kinase 3 (ADK3), une enzyme contenant une région intrinsèquement désordonnée (IDR). Cette enzyme a été isolée par notre approche globale pour caractériser les IDPs de l’algue verte. L’extension C-terminale désordonnée (CTE) de cette enzyme lui confère de nouvelles fonctions comme par exemple, la formation d’un complexe bi-enzymatique avec la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH), la régulation (négative) de l'activité GAPDH avec le NADPH comme cofacteur, et le rôle de chaperon pour la GAPDH en la protégeant de la dénaturation par traitement thermique et de l’agrégation
The objectives of this work were to bring a conceptual breakthrough for an in-depth understanding of the molecular mechanisms of intrinsically disordered proteins (IDPs) and their roles in the cellular physiology of Chlamydomonas reinhardtii. Using experimental approaches, 682 heat-resistant proteins were identified as putative IDPs. Among them, 299 proteins were consistently predicted as IDPs by all four disordered predictors. The mean percentage of disordered residues content of these IDPs is about 20%, and most of them (~70%) are addressed to other compartments than mitochondrion and chloroplast. These newly identified IDPs from C. reinhardtii have a biased amino acid composition as regard to other IDPs from the Database of protein disorder (DisProt). Furthermore, they play diverse molecular functions, and 54% of them are the targets for phosphorylation. Our work also revealed more knowledge of the IDR-containing protein adenylate kinase 3 (ADK3) that was extracted by heat-treatment. Its disordered C-terminal extension (CTE) brought new functions to this protein. For instance, via its CTE, ADK3 can form a bi-enzyme complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), down-regulates the NADPH-dependent GAPDH activity, and behaves as a chaperone for GAPDH against its aggregation and inactivation under heat-treatment

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4

Rutter, Gil. "Coarse-grained simulations of intrinsically disordered peptides". Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/74165/.

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Intrinsically disordered proteins (IDPs) are functional proteins which lack a unique and stable tertiary structure. IDPs such as n16N are involved in biomineralisation, the process by which organisms produce mineral materials, such as shells. Here, the role that accelerated simulation can play in the study of IDPs is examined and furthered. The coarse-grained models PLUM and PRIME20 are implemented and refined based on existing single-chain n16N simulations. In conjunction with the replica exchange molecular dynamics technique, the models are used to simulate systems of 1, 2, 3 and 6 chains of n16N, and a mutant form n16NN. The modified PLUM model is in striking agreement with existing hypotheses regarding the structure of n16N, when simulations are run in multiplicity. The PRIME20 model has difficulty producing plausible backbone structure in every system size, though it does fulfil some expectations regarding residue interaction specificity. New hypotheses are offered on bulk n16N and n16NN aggregation based on the presented data. Future directions for development of accelerated simulation techniques for IDPs are suggested.

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5

Rogers, Joseph Matthew. "Coupled folding and binding of intrinsically disordered proteins". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648368.

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6

Troilo, Francesca. "Molecular mechanisms of folding of intrinsically disordered proteins". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0755.

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Cette thèse étudie les mécanismes moléculaires du repliement des protéines intrinsèquement désordonnées (IDPs). En particulier, nous avons étudié l'interaction entre NTAIL, le domaine intrinsèquement désordonné de la nucléoprotéine du virus de la rougeole, et le domaine X (XD) de la phosphoprotéine. Lors de l’interaction avec XD, NTAIL subit un repliement α-hélical au sein d’un élément de reconnaissance moléculaire (α-MoRE), alors que les régions adjacentes restent désordonnées («fuzzy»). L’appendice «fuzzy» précédant l'α-MoRE diminue l’affinité pour XD ainsi que la vitesse de repliement de l'α-MoRE. A l’aide de nombreux variants de NTAIL (variants ponctuels, tronqués et artificiels) et d’expériences de cinétique fondées sur la technique de relaxation par saut de température, nous avons montré que NTAIL se replie via un mécanisme de nucléation-condensation, la partie centrale de l'hélice étant responsable des interactions initiales. Nous avons aussi montré que la région «fuzzy» réduit la vitesse de repliement de l'α-MoRE via une combinaison d'effets entropiques et enthalpiques. L’étude de l'interaction entre NTAIL et un variant de XD qui adopte uniquement la conformation native, a montré que les étapes de liaison et de repliement de NTAIL dépendent fortement de la forme de XD, suggérant que cette IDP se replie par nucléation hétérogène via un mécanisme induit par la forme du partenaire («templated folding»). Enfin, nous avons également étudié l'interaction entre NTAIL et la protéine de choc thermique de 70 kDa (Hsp70). Des approches mutationnelles ont montré que NTAIL lie Hsp70 et XD en utilisant différents mécanismes, confirmant ainsi l'extrême polymorphisme des IDPs
In this thesis, we studied the interaction between the intrinsically disordered domain of the nucleoprotein (N) of Measles virus (MeV), NTAIL, and its partner XD, the X domain of the MeV phosphoprotein (P). It had been previously shown that the α-MoRE (residues 489-506) of NTAIL undergoes an α-helical folding after binding to XD (induced fit mechanism) while regions flanking the α-MoRE remain disordered (fuzzy) in the complex. The fuzzy appendage preceding the α-MoRE was shown to decrease the binding affinities towards XD and the rate of folding of the α-MoRE. In this thesis, by producing NTAIL variants (single-site variants, truncation variants, artificial variants) and performing kinetic experiments of the interaction with XD, we studied the folding after binding mechanism of NTAIL at the single residue level, and investigated the mechanisms through which the fuzzy region hampers the binding affinity and the folding rate of the α-MoRE. We concluded that the central part of the helix is responsible for the initial interactions driving the binding with XD. Moreover, we found that the fuzzy region causes a decrease in the folding rate of the α-MoRE through a combination of entropic and enthalpic effects. We also studied the interaction between NTAIL and a variant of XD, I504A, that populates only the native state. These studies showed that both the binding and the folding steps of the NTAIL-XD interaction are highly dependent on the shape of XD, suggesting that this IDP folds by heterogeneous nucleation via a mechanism induced by the shape of the partner (templated folding)

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7

Bagheri, Mehran. "Intrinsically Disordered Proteins: Mechanics, Assemblies, and Structural Transitions". Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36576.

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Proteins are essential parts of living organisms that initiate and control almost all cellular processes. Despite the widely accepted belief that all functional proteins fold into stable and well-defined three-dimensional (3D) structures mandatory for protein activity, the existence of biologically functional disordered proteins has been increasingly recognized during past two decades. Proteins with inherent structural disorder, commonly known as intrinsically disordered proteins (IDPs), play many roles in a biological context. However, in contrast to their folded counterparts, they are dynamically unstructured and typically fluctuate among many conformations even while performing biological functions. In fact, it is this dynamical structural heterogeneity that that allows for IDPs to interact with other biological macromolecules in unique ways. Moreover, while a majority of proteins in eukaryotic proteomes have been found to have intrinsically disordered regions (IDR), the mechanisms by which protein disorder fives rise to biological functionality is still not well understood. Through a series of simulation studies on specific systems, this thesis probes several aspects of the emerging structure-function paradygm of IDPs, namely the mechanics, intermolecular assembly, and structural transitions occurring in these proteins. The lack of well-defined 3D structure in IDPs gives rise to distinct mechanical properties, the subject of the first study in the thesis on the elasticity of a elastomeric gluten-mimetic polypeptide with an intrinsically disordered character. This disordered polypeptide was shown to exhibit distinctively variable elastic response to a wide range of tensions, which a classical worm-like chain model failed to accurately describe, thus requiring a molecular-level analysis. IDPs frequently are frequently involved in protein-protein interactions, the focus of the second study on the propensity of an IDR, the B domain in dynamin-related protein 1 (Dpr1), to self-assemble into dimer structures while remaining disordered in all solution conditions. Despite a hypothesized auto-inhibitory role for this domain in Dpr1 that was assumed to be triggered by an disordered-to-order transition, the B domains in solution showed no tendency to form ordered structures even in the presence of order promoting osmolytes. Instead, self-association in the presence of osmolyte was found to occur by favorable intermolecular intereactions between specific region on the surface of the B-domains. Other IDPs do undergo a disorder-to-order transition in response to environmental cues, in ways that are unique disordered proteins, the focus of the last study on intermolecular ordering transitions in silk-like proteins. Factors such as protein sequence and physical tension were investigated, and results suggested that tyrosine residues in the key silk sequence motifs promote templating of beta structure from disordered precursors and that elongational stresses preferentialy stabilize antiparallel beta-sheet order. Together, these three computational studies provide insight into the nature of the structure-function mechanisms of IDPs.

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8

Midic, Uros. "Genome-Wide Prediction of Intrinsic Disorder; Sequence Alignment of Intrinsically Disordered Proteins". Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/159800.

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Computer and Information Science
Ph.D.
Intrinsic disorder (ID) is defined as a lack of stable tertiary and/or secondary structure under physiological conditions in vitro. Intrinsically disordered proteins (IDPs) are highly abundant in nature. IDPs possess a number of crucial biological functions, being involved in regulation, recognition, signaling and control, e.g. their functional repertoire complements the functions of ordered proteins. Intrinsically disordered regions (IDRs) of IDPs have a different amino-acid composition than structured regions and proteins. This fact has been exploited for development of predictors of ID; the best predictors currently achieve around 80% per-residue accuracy. Earlier studies revealed that some IDPs are associated with various human diseases, including cancer, cardiovascular disease, amyloidoses, neurodegenerative diseases, diabetes and others. We developed a methodology for prediction and analysis of abundance of intrinsic disorder on the genome scale, which combines data from various gene and protein databases, and utilizes several ID prediction tools. We used this methodology to perform a large-scale computational analysis of the abundance of (predicted) ID in transcripts of various classes of disease-related genes. We further analyzed the relationships between ID and the occurrence of alternative splicing and Molecular Recognition Features (MoRFs) in human disease classes. An important, never before addressed issue with such genome-wide applications of ID predictors is that - for less-studied organisms - in addition to the experimentally confirmed protein sequences, there is a large number of putative sequences, which have been predicted with automated annotation procedures and lack experimental confirmation. In the human genome, these predicted sequences have significantly higher predicted disorder content. I investigated a hypothesis that this discrepancy is not correct, and that it is due to incorrectly annotated parts of the putative protein sequences that exhibit some similarities to confirmed IDRs, which lead to high predicted ID content. I developed a procedure to create synthetic nonsense peptide sequences by translation of non-coding regions of genomic sequences and translation of coding regions with incorrect codon alignment. I further trained several classifiers to discriminate between confirmed sequences and synthetic nonsense sequences, and used these predictors to estimate the abundance of incorrectly annotated regions in putative sequences, as well as to explore the link between such regions and intrinsic disorder. Sequence alignment is an essential tool in modern bioinformatics. Substitution matrices - such as the BLOSUM family - contain 20x20 parameters which are related to the evolutionary rates of amino acid substitutions. I explored various strategies for extension of sequence alignment to utilize the (predicted) disorder/structure information about the sequences being aligned. These strategies employ an extended 40 symbol alphabet which contains 20 symbols for amino acids in ordered regions and 20 symbols for amino acids in IDRs, as well as expanded 40x40 and 40x20 matrices. The new matrices exhibit significant and substantial differences in the substitution scores for IDRs and structured regions. Tests on a reference dataset show that 40x40 matrices perform worse than the standard 20x20 matrices, while 40x20 matrices - used in a scenario where ID is predicted for a query sequence but not for the target sequences - have at least comparable performance. However, I also demonstrate that the variations in performance between 20x20 and 20x40 matrices are insignificant compared to the variation in obtained matrices that occurs when the underlying algorithm for calculation of substitution matrices is changed.
Temple University--Theses

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9

Herrera, Nieto Pablo 1992. "A Study of intrinsically disordered proteins using molecular dynamics simulations". Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/670061.

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Over the last decades molecular dynamics simulations have been successfully applied to relevant biological problems such as protein-ligand, protein-protein binding as well as protein folding. A perfect challenge for molecular simulations is the study of intrinsically disordered proteins as they present faster timescales than structured proteins, which can be explored more exhaustively. The main objectives of this work are the exploration of the conformational space of p53 by revealing the presence of many partially ordered states, the reconstruction of the coupled folding and binding of a disordered protein and its folded partner by applying novel reinforcement learning inspired sampling algorithms, and the performance of free-ligand binding assays in order to address the potential druggability of disordered proteins. The compendium of work presented here contributes to the understanding of such intrinsically disordered proteins at an atomistic level, highlighting key aspects of their behaviour in isolation, binding mechanisms, and external modulation.
A lo largo de las últimas décadas las simulaciones de dinámica molecular han sido aplicadas éxitosamente en problemas biológicos como la interación proteína-ligando o proteína-proteína así como plegamiento de proteínas. Un desafío idóneo para las simulaciones es el estudio de proteína desordenadas, ya que presentan escalas de tiempo mas rápidas que la proteínas plegadas, permientiendo una exploración mas exhaustiva de las mismas. Entre los principales objetivos del presente trabajo figuran la exploración del paisaje conformacional de p53 revelando la presencia de estados parcialmente ordenados, la reconstrucción del acoplamiento de unión y plegamiento de una proteína desordenada a su pareja, aplicando novedosos algoritmos de muestreo inspirados en aprendizaje reforzado, y la realización de ensayos de unión de ligando libre para abordar la potencial drogabilidad de las proteínas desordenadas. El compendio del trabajo presentado aquí contribuye a entnder dichas proteínas a nivel atómico, destacando aspectos clave de su comportamiento en aislamiento, mecanismos de unión, y su modulación externa.

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10

Miljak, Marija. "Conformational sampling of intrinsically disordered peptides by enhanced sampling methods". Thesis, University of Southampton, 2017. https://eprints.soton.ac.uk/422232/.

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The aim of this study was to explore the conformational equilibrium of four cyclic hormone peptides in order to investigate to what extent the bound conformational state can be observed from the solution phase simulations. The studied cyclic peptides share the same structural motif of a six membered ring closed by disulphide bridge between the cysteine residues. They also belong to the class of intrinsically disordered peptides known to exist in an equilibrium of different conformations. Elucidating their conformational ensemble using traditional experimental techniques has proven hard due to the fast interconversion between conformational states, and thus molecular dynamics simulation may help in providing a detailed picture of the peptide’s conformational ensemble. However, conventional molecular dynamics simulation are limited by the long time scale required to observe many conformational motions. Therefore in this work Replica Exchange techniques were applied to test the rate of convergence in conformational sampling. Moreover, to predict the conformational equilibrium of the peptides, a combination of results from enhanced sampling methods, DFT calculations and NMR experiments was used. It was found that calculated chemical shifts weighted by the ensemble populations of each conformational state were better able to reproduce the experimental chemical shift data, over and above any single peptide conformation. This result supports the use of enhanced sampling molecular dynamics computer simulations to study intrinsically disordered peptides. The knowledge of the conformational equilibrium and the relative populations of the unbound states of the peptides obtained using this approach may help in predicting the structural and functional roles of the bound state peptide. Another purpose of this work was also to check the extent to which a difference in peptide sequence may contribute to their functional diversity. Finally, the performance of the Replica Exchange simulations was compared, indicating that Solute Tempering is to be preferred over temperature Replica Exchange for reasons of computational efficiency.

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11

Ullman, Orly. "Modeling intrinsically disordered proteins ; a comprehensive study of [alpha]-synuclein". Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97985.

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Thesis: Ph. D. in Physical Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.
Cataloged from PDF version of thesis. In title on title page, "[alpha]" appears as lower case Greek letters. Vita.
Includes bibliographical references (pages 91-104).
Parkinson's disease (PD) affects over 10 million people worldwide and has no cure. Moreover, current treatments for PD have limited efficacy. Studies that advance our understanding of the mechanism of neurodegeneration in PD will provide guidance in our search for effective therapies for this neurodegenerative disorder. PD is characterized clinically by motor deficits - namely resting tremors, rigidity, bradykinesia and postural instability - and pathologically by intraneuronal inclusions in the substantia nigra. Several studies suggest that a-synuclein, the major component of these intracellular inclusions, plays a major role in the neurodegenerative process. Therefore understanding the structural properties of [alpha]-synuclein and its aggregation mechanism is of particular interest. [alpha]-synuclein is particularly challenging to study because it is an Intrinsically Disordered Protein (IDP); i.e., it lacks a well-defined structure in aqueous solution. Unlike folded proteins, IDPs typically interconvert between many different conformations during their biological lifetime. In this thesis we apply novel methods to develop models for IDPs and apply them to asynuclein. The overriding hypothesis that forms the basis of this work is that IDPs in solution can be modeled as a finite set of energetically favorable structures, where each structure corresponds to an energy minimum on a complex energy landscape. The number of structures in the resulting ensemble is related to the resolution in which one wishes to view the energy landscape of the protein. We demonstrate that this approach leads to new insights into the aggregation mechanism of [alpha]-synuclein.
by Orly Ullman.
Ph. D. in Physical Chemistry

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12

Reddy, Krishna D. "Regulation of Palmitoylation Enzymes and Substrates by Intrinsically Disordered Regions". Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6572.

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Protein palmitoylation refers to the process of adding a 16-carbon saturated fatty acid to the cysteine of a substrate protein, and this can in turn affect the substrate’s localization, stability, folding, and several other processes. This process is catalyzed by a family of 23 mammalian protein acyltransferases (PATs), a family of transmembrane enzymes that modify an estimated 10% of the proteome. At this point in time, no structure of a protein in this family has been solved, and therefore there is poor understanding about the regulation of the enzymes and their substrates. Most proteins, including palmitoylation enzymes and substrates, have some level of intrinsic disorder, and this flexibility can be important for signaling processes such as protein- protein interactions and post-translational modifications. Therefore, we assumed that examining intrinsic disorder in palmitoylation enzymes and substrates would yield insight into their regulatory mechanisms. First, we found that among other factors, utilizing intrinsic disorder predictions led to a palmitoylation predictor that significantly outperformed existing predictors. Next, we discovered a conserved region of predicted disorder-to-order transition in the disordered C-termini of the PAT family. In Erf2, the yeast Ras PAT, we developed a model where this region reversibly interacts with membranes, and we found that this region mediates interaction with Acc1, an enzyme involved in fatty acid metabolism processes. Finally, we found that an XLID-associated nonsense mutation in zDHHC9, the mammalian Ras PAT, removed a disordered region that was critical for enzyme localization. Future studies of palmitoylation utilizing the framework of intrinsic disorder may lead to additional insights about this important regulatory process.

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13

Norris, Nicole Catherine. "Structural and functional studies of intrinsically disordered fibronectin-binding proteins". Thesis, University of York, 2009. http://etheses.whiterose.ac.uk/570/.

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Bacterial fibronectin-binding proteins (FnBPs) mediate adhesion of bacteria to host tissues through binding to the human protein fibronectin (Fn). FnBPs are predicted to contain a series of intrinsically disordered Fn-binding repeats (FnBRs), which undergo a disorder-to-order transition on binding up to five F1-modules (1−5F1) from the N terminal domain of Fn through a tandem β-zipper interaction. This work investigated these predictions for SfbI-5, an FnBR from the important human pathogen Streptococcus pyogenes. SfbI-5 was shown to behave as an intrinsically disordered protein (IDP), with no stable secondary or tertiary structure. The C terminal third of SfbI 5 was shown to have propensity for β-strand conformations and, thus, might contain a preformed contact site involved in the initial interaction between SfbI-5 and 1−5F1. A high resolution structure of an SfbI-5 peptide in complex with 2F13F1 demonstrated that peptide binding occurs through formation of a tandem β-zipper with the concomitant formation of a large, extended intermolecular interface. Conserved FnBR residues play roles similar to those played by residues in related complexes involving Staphylococcus aureus FnBRs. The effect of mutating these residues on SfbI-5 binding to 1−5F1 was shown to be modest, suggesting that large, extended interfaces, when formed by IDPs, might be tolerant to mutations in the IDP. A putative FnBR, TickFnBR, was identified in a salivary protein from the blood-feeding tick Ixodes scapularis, which is a vector for human diseases. TickFnBR was shown to bind to 1−5F1 and also to Fn in human plasma. A high resolution structure demonstrated that a TickFnBR peptide binds to 2F13F1 through a tandem β-zipper. TickFnBR is the first eukaryotic FnBR to be identified, suggesting the tandem β zipper interaction is a useful mechanism for harmful organisms to target and exploit human Fn.

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Patel, Avnish. "Studies of the intrinsically disordered N-terminus of murine prion protein". Thesis, University of Reading, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577771.

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Mammalian prion protein is able to cause a multitude of neurological maladies, most notably the transmissible spongiform echephalopathies. All are characterised by the misfolding of a non-pathogenic form the cellular protein Prpc to a misfolded aggregated isoform Prpscwhich leads to neurotoxicity and apoptosis characterised by brain spongiosis and amyloid plaque deposits. The exact molecular mechanisms of pathogenicity remain unknown for both the transmissible and inherited forms of disease. An artificially generated deletion of residues 105-125 of Prpc, encoding largely hydrophobic amino acids, in the intrinsically disordered N-terminus of the protein is hig~ly neurotoxic and has been shown to mediate a TSE like phenotype when expressed in transgenic mice. A possible mechanism of action consistent with prion protein exhibiting varying isoforms is that the deletion mutant may fold to a structure comparative to that of a naturally occurring pathogenic form. Biochemical and biophysical characterisation of the deleted form of Prpc could support this possibility. To explore the role of residues 95-135 in protein folding an extended set of deletion mutants of the hydrophobic region were created and the encoded Prpc like proteins expressed and purified. Among the mutants made it was found that deletion of solely residues 105-125 within the hydrophobic region gave rise to subtle structural alterations when compared to parental Prpc. As Prpc is a GPI anchored protein normally associated with a membrane environment, membrane interaction assays were also performed with deletion mutants revealing that that a charge cluster and hydrophobic region acted synergistically to bind, insert into and disrupt anionic membranes. These findings did not support a mechanism of action for the deletion ~105-125 mediating toxicity through membrane pore formation but rather a subtle structural change that may be consistent with a receptor 4 mediated toxicity model. Possible further cellular and structural studies with defined deletions of Prpc with a view to deciphering the neurotoxic mode of action are discussed. 5

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15

Borcherds, Wade Michael. "Structure, Dynamics, and Evolution of the Intrinsically Disordered p53 Transactivation Domain". Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4640.

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in numerous disease states, including cancers and neurodegenerative diseases. All proteins are dynamic in nature, occupying a range of conformational flexibilities. This inherent flexibility is required for their function, with ordered proteins and IDPs representing the least flexible, and most flexible, respectively. As such IDPs possess little to no stable tertiary or secondary structure, they instead form broad ensembles of heterogeneous structures, which fluctuate over multiple time scales. Although IDPs often lack stable secondary structure they can assume a more stable structure in the presence of their binding partners in a coupled folding binding reaction. The phenomenon of the dynamic behavior of IDPs is believed to confer several functional advantages but remains poorly understood. To that end the dynamic and structural properties of a family of IDPs - p53 transactivation domains (TAD) was measured and compared with the sequence divergence. Interestingly we were able to find stronger correlations between the dynamic properties and the sequence divergence than between the structure and sequence, suggesting that the dynamic properties are the primary trait being xiii conserved by evolution. These correlations were strongest within clusters of the IDPs that correlated with known protein binding sites. Additionally, we show strong correlations between the several available disorder predictors and the backbone dynamics of this family of IDPs. This indicates the potential of predicting the dynamic behavior of proteins, which may be beneficial in future drug design. The limited number of atomic models currently determined for IDPs hampers understanding of how their amino acid sequences dictate the structural ensembles they adopt. The current dearth of atomic models for IDPs makes it difficult to test the following hypotheses: 1. The structural ensembles of IDPs are dictated by local interactions. 2. The structural ensembles of IDPs will be similar above a certain sequence identity threshold. Based on the premise that sequence determines structure, structural ensembles were determined and compared for a set of homologous IDPs. Utilizing orthologues allows for the identification of important structural features and behaviors by virtue of their conservation. A new methodology of creating ensembles was implemented that broadly samples conformational space. This allowed us to find recurring local structural features within the structural ensembles even between the more distantly related homologues that were processed. This method of ensemble creation is also the first method to show convergence of secondary structural characteristics between discrete ensembles.

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16

Ramberger, Evelyn. "Spatial protein interaction networks of the intrinsically disordered transcription factor CEBPA". Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21840.

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Der Transkriptionsfaktor CEBPA reguliert Differenzierung und Proliferation in verschiedenen Zelltypen und spielt eine herausragende Rolle in der Hämatopoese. Die CEBPA RNA kann in die lange P42-Isoform oder die N-terminal verkürzte P30-Isoform translatiert werden. Während P42-CEBPA differenzierungsinduzierend wirkt, ist P30 als Inhibitor von P42 und als Onkogen in akuter myeloider Leukämie beschrieben. Die Modularität und Multifunktionalität von CEBPA, die ihn zahlreichen Studien beobachtet wurde, lässt sich möglicherweise durch differentielle Protein–Protein-Interaktionen erklären. Zahlreiche post-translationale Modifikationen (PTMs) und die intrinsisch ungeordnete, flexible Struktur von CEBPA stellen jedoch eine Herausforderung für traditionelle Ansätze in Proteininteraktionsstudien dar. In der vorliegenden Arbeit wird ein neuer, alternativer Ansatz präsentiert, der auf einem in vitro Proteininteraktions-screen auf einer Peptidmatrix (PRISMA) und Biotinligase proximity labelling (BioID) in lebenden Zellen basiert. In einem PRISMA-screen wurden 120 CEBPA Peptide auf Proteininteraktionen mit Proteinextrakt aus myeloiden Zellen untersucht. Im Screen wurden 40 verschiedene CEBPA PTMs inkludiert, unter anderem auch die hier erstmals neu beschriebenen Methylierungen der CEBPA Argininreste R12 und R142. Daten aus dem PRISMA-screen wurden mit BioID Experimenten in myeloiden Zellen validiert, um eine Proteininteraktionslandkarte von CEBPA zu generieren, die 52 bekannte und 68 neue CEBPA Proteininteraktoren umfasst. Hotspots für Proteininteraktionen fallen in evolutionär konservierte CEBPA Regionen und der Vergleich des Bindungsprofils mit publizierten Daten zeigt Ähnlichkeiten zu verwandten Transkriptionsfaktoren der CEBP Familie. Die Ergebnisse legen nahe, dass die Multifunktionalität von CEBPA von multivalenten Proteininteraktionen in Abhängigkeit von PTMs koordiniert wird, um CEBPA mit dem epigenetischen und transkriptionellen Apparat der Zelle verknüpfen.
The pioneering transcription factor CEBPA plays a lineage-instructing role during haematopoiesis and also regulates proliferation and differentiation in many other cell types. The CEBPA RNA can be translated into a full length (P42-CEBPA) or N-terminally truncated isoform (P30-CEBPA). While P42 induces differentiation in various cell types, the P30 isoform is mostly regarded as a dominant inhibitor of P42-CEBPA and acts as an oncogene in acute myeloid leukaemia. Protein interactions may be the key to explaining the functional plasticity and modularity of CEBPA that has been demonstrated in diverse experimental settings. However, the disordered structure and the numerous post-translational modification sites (PTMs) of CEBPA pose a challenge to traditional protein interaction studies. In the present work, a novel alternative approach is presented that combines an in vitro protein interaction screen on a peptide matrix (PRISMA) with biotin ligase proximity labelling (BioID) in living cells. To this end, 120 CEBPA peptides were probed for protein interactions with PRISMA. The screen comprised 40 different PTMs, including newly identified CEBPA arginine methylation sites. PRISMA data was validated with BioID experiments and generated a detailed CEBPA protein interaction map in myeloid cells. The interactome presented here contains 52 known and 68 novel CEBPA interactors that can now be mapped across the CEBPA sequence in a PTM dependent fashion. Hotspots of protein interaction correlated with conserved regions and comparison with previously published data revealed related binding profiles of homologous CEBP regions. Taken together, the data indicates that the functional plasticity of CEBPs is orchestrated by multivalent protein interactions and PTMs to configure a dynamic CEBP hub that interacts with many partners of the transcriptional and epigenetic machinery.

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17

Estana, Alejandro. "Algorithms and comptutational tools for the study of Intrinsically Disordered Proteins". Thesis, Toulouse, INSA, 2020. http://www.theses.fr/2020ISAT0012.

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Les protéines intrinsèquement désordonnées (IDPs) sont impliquées dans de nombreux processus biologiques. Leur plasticité facilite des tâches très spécialisées dans la régulation et la signalisation cellulaires, et leur dysfonctionnement est lié à des pathologies graves. Comprendre les rôles fonctionnels des IDPs nécessite leur caractérisation structurale, qui est extrêmement difficile, utilisant un couplage entre méthodes expérimentales et informatiques. Contrairement aux protéines globulaires, les IDPs ne peuvent pas être représentés par une seule conformation et leurs modèles doivent être basés sur des ensembles de conformations représentant une distribution des états que la protéine adopte en solution. Alors que les régions purement destructurées peuvent être bien modélisés par les outils de bioinformatique disponibles, ces outils ne parviennent pas à reproduire l'équilibre conformationnel présent dans des régions partiellement structurées.Dans cette thèse, nous proposons plusieurs méthodes bioinformatiques qui, combinées avec des données expérimentales, permettent une meilleure caractérisation structurale des IDPs. Elles peuvent être regroupées en deux catégories : les méthodes de construction de modèles d'ensembles conformationnels, et les méthodes de simulation de transitions conformationnelles.Avec le premier type de méthodes, nous proposons une nouvelle approche qui améliore les méthodes existantes afin de générer des ensembles conformationnels réalistes, en permettant de reproduire les régions partiellement structurées dans les IDPs.Cette méthode exploite les informations structurales codées dans une base de données de fragments de trois résidus (tripeptides) extraites de structures protéiques à haute résolution.Nous avons montré que les ensembles conformationnels générés par notre méthode reproduisent avec précision les descriptions structurales obtenues à partir d’expériences de RMN et SAXS pour neuf IDPs de référence. Exploitant également la base de données des tripeptides, nous avons développé un algorithme pour prédire la propension de certaines régions dans des IDPs à former des éléments de structure secondaire. Cette nouvelle méthode fournit des résultats plus précis que ceux de prédicteurs plus couramment utilisés, pour nos IDPs de référence.Avec le second type de méthodes, nous avons développé une approche originale pour modéliser le mécanisme de repliement des éléments de structure secondaire. Le calcul des transitions est formulé comme un problème de recherche de chemin discret utilisant la base de données de tripeptides. Pour évaluer l'approche, nous l'avons appliquée à deux petits polypeptides synthétiques imitant deux motifs structuraux communs. Les mécanismes de repliement extraits sont très similaires à ceux obtenus par des approches traditionnelles coûteuses en calcul. Enfin, nous avons développé une méthode plus générale pour calculer les chemins de transition entre conformations dans un ensemble d'IDPs. Elle repose sur une variante multi-arbres de l'algorithme TRRT, développé au LAAS-CNRS, et qui a donné de bons résultats pour de petites et moyennes biomolécules. Afin d'appliquer cette méthode aux IDPs, nous avons proposé une stratégie hybride pour la parallélisation de l'algorithme, permettant une exécution efficace dans des clusters.Outre les travaux méthodologiques mentionnés, j'ai participé activement à des travaux multidisciplinaires avec des biophysiciens et des biologistes, en appliquant ces méthodes à l'étude de systèmes biologiques importants, comme la protéine huntingtine impliquée dans la maladie de Huntington.En conclusion, les travaux menés au cours de ma thèse ont permis de mieux comprendre la relation séquence-structure des IDPs, ouvrant la voie à de nouvelles applications. En effet, ceci nous permettra, par exemple, d’anticiper les perturbations structurales dues aux mutations et, in fine, à la conception rationnelle d’IDPs spécifiques pour des applications biotechnologiques
Intrinsically Disordered Proteins (IDPs) are involved in many biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional roles of IDPs requires their structural characterization, which is extremely challenging, and needs a tight coupling of experimental and computational methods. In contrast to structured/globular proteins, IDPs cannot be represented by a single conformation, and their models must be based on ensembles of conformations representing a distribution of states that the protein adopts in solution. While purely random coil ensembles can be reliably constructed by available bioinformatics tools, these tools fail to reproduce the conformational equilibrium present in partially-structured regions.In this thesis, we propose several computational methods that, combined with experimental data, provide a better structural characterization of IDPs. These methods can be grouped in two main categories: methods to construct conformational ensemble models, and methods to simulate conformational transitions.Contributing to the first type of methods, we propose a new approach to generate realistic conformational ensembles that improves previously existing methods, being able to reproduce the partially-structured regions in IDPs.This method exploits structural information encoded in a database of three-residue fragments (tripeptides) extracted from high-resolution experimentally-solved protein structures.We have shown that conformational ensembles generated by our method reproduce accurately structural descriptors obtained from NMR and SAXS experiments for a benchmark set of nine IDPs. Also exploiting the tripeptide database, we have developed an algorithm to predict the propensity of some fragments inside IDPs to form secondary structure elements. This new method provides more accurate results than those of the most commonly-used predictors available on our benchmark set of well-characterized IDPs.Contributing to the second type of methods, we have developed an original approach to model the folding mechanism of secondary structural elements. The computation of conformational transitions is formulated as a discrete path search problem using the tripeptide database. To evaluate the approach, we have applied the strategy to two small synthetic polypeptides mimicking two common structural motifs in proteins. The folding mechanisms extracted are very similar to those obtained when using traditional, computationally expensive approaches. Finally, we have developed a more general method to compute transition paths between a (possibly large) set of conformations of an IDP. This method builds on a multi-tree variant of the TRRT algorithm, developed at LAAS-CNRS, and which provided good results for small and middle-sized biomolecules. In order to apply this method to IDPs, we have proposed a hybrid strategy for the parallelization of the algorithm, enabling an efficient execution in computer clusters.In addition to the aforementioned methodological work, I have been actively involved in multidisciplinary work, together with biophysicists and biologists,where I have applied these methods to the investigation of important biological systems, in particular the huntingtin protein, the causative agent of Huntington's disease.In conclusion, the work carried out during my PhD thesis has enabled a better understanding of the relationship between sequence and structural properties of IDPs, paving the way to novel applications. For example, this deeper understanding of sequence-structure relationships will enable us to anticipate structural perturbations exerted by sequence mutations, and subsequently, the rational design of IDPs with tailored properties for biotechnological applications

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18

Ruzmetov, Talant A. "THE ROLE OF CHAIN FLEXIBILITY AND CONFORMATIONALDYNAMICS ON INTRINSICALLY DISORDERED PROTEINASSOCIATION". Kent State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=kent1564588247414425.

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19

Kragelj, Jaka. "Structure and dynamics of intrinsically disordered regions of MAPK signalling proteins". Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV060/document.

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Les voies de transduction du signal cellulaire permettent aux cellules de répondre aux signaux de l'environnement et de les traiter. Les voies de transduction de kinases MAP (MAPK) sont bien conservées dans toutes les cellules eucaryotes et sont impliquées dans la régulation de nombreux processus cellulaires importants. Les régions intrinsèquement désordonnées (RID), présentes dans de nombreuses MAPK, n'étaient pas encore structurellement caractérisées. Les RID de MAPK sont particulièrement importantes car elles contiennent des motifs de liaison qui contrôlent les interactions entre les protéines MAPK elles-mêmes et aussi entre les protéines MAPK et d'autres protéines contenant les mêmes motifs. La résonance magnétique nucléaire (RMN) en combinaison avec d'autres techniques biophysiques a été utilisée pour étudier les RID de kinase des voies de transduction du signal MAPK. La spectroscopie RMN est bien adaptée pour l'étude des protéines intrinsèquement désordonnées à l'échelle atomique. Les déplacements chimiques et couplages dipolaires résiduels peuvent être utilisés conjointement avec des méthodes de sélection d'ensemble pour étudier la structure résiduelle dans les RID. La relaxation de spin nucléaire nous renseigne sur les mouvements rapides. Des titrations par RMN et des techniques de spectroscopie d'échange peuvent être utilisées pour surveiller la cinétique d'interactions protéine-protéine. Cette étude contribuera à la compréhension du rôle des RID dans les voies de transduction du signal cellulaire
Protein signal transduction pathways allow cells respond to and process signals from the environment. A group of such pathways, called mitogen-activated protein kinase (MAPK) signal transduction pathways, is well conserved in all eukaryotic cells and is involved in regulating many important cell processes. Long intrinsically disordered region (IDRs), present in many MAPKs, have remained structurally uncharacterised. The IDRs of MAPKs are especially important as they contain docking-site motifs which control the interactions between MAPK proteins themselves and also between MAPKs and other interacting proteins containing the same motifs. Nuclear magnetic resonance (NMR) spectroscopy in combination with other biophysical techniques was used to study IDRs of MAPKs. NMR spectroscopy is well suited for studying intrinsically disordered proteins (IDPs) at atomic-level resolution. NMR observables, such as for example chemical shifts and residual dipolar couplings, can be used together with ensemble selection methods to study residual structure in IDRs. Nuclear spin relaxation informs us about fast pico-nanosecond motions. NMR titrations and exchange spectroscopy techniques can be used to monitor kinetics of protein-protein interactions. The mechanistic insight into function of IDRs and motifs will contribute to understanding of how signal transduction pathways work

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20

Ozdilek, Bagdeser A., Valery F. Thompson, Nasiha S. Ahmed, Connor I. White, Robert T. Batey y Jacob C. Schwartz. "Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding". OXFORD UNIV PRESS, 2017. http://hdl.handle.net/10150/625471.

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RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA.

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21

Maffei, Mariano. "Structural and functional characterization of the intrinsically disordered Unique domain of c-Src". Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/285298.

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Objectives: Objective 1 – Effect of mutations perturbing the ULBR of the Unique domain of c-Src. The first objective of this thesis was the in vitro characterization of the effect of mutations in the ULBR on the previously determined main interactions of the Unique domain: binding to lipids and to the SH3 domain. Objective 2 - Phosphorylation of c-Src Unique domain in Xenopus laevis oocytes and mammalian cell extracts. Phosphorylation of the Unique domain of c-Src is one of the modulation parameters affecting lipid binding and had previously been reported to affect c-Src activity. The second objective was to study the phosphorylation of the isolated Unique domain in Xenopus laevis oocytes, that enabled the study of phosphorylation in a living cell and in cell extracts, that allowed further manipulation of the phosphatase/kinase network. Objective 3 - Functional studies of the Unique domain in the context of the full-length protein. The last objective was the evaluation of the functional significance of the modifications investigated in the isolated Unique domain as objectives 1 and 2 in a human colorectal cell line and in the context of the full length protein. The present thesis addressed fundamental aspects of the biological role of the intrinsically disordered Unique domain of c-Src that until now was poorly understood. We have first characterized through a structural biology approach the effects of sitedirected ULBR mutations on the previously described interactions with lipids and with the folded SH3 domain. Then, we have investigated the phosphorylation events taking place on the Unique domain by state-of-the-art in-cell NMR techniques. Finally, we have shown the functional role of USrc in the context of normal and cancer cell lines. All together, our results have demonstrated that the Unique domain of human c-Src actively participates in the regulation mechanism in which the tyrosine kinase c-Src is involved. This study represents a new essential milestone in the understanding of how c-Src can control a wide variety of signaling events inside cells. Furthermore, it potentially opens the way to the possibility of new applications for cancer therapy through the development of novel selective drugs targeting functional active residues locating in the Unique domain. This thesis represents a “proof-of-concept” of how from the atomistic comprehension of a subject of interest (structural approach) it can be possible to understand and successively demonstrate the biological significance of the observed phenomena (functional approach). Conclusions: - Mutations in the Unique Lipid Binding Region affect the lipid binding capability of USrc but not the inter-domain interaction with the folded SH3. - Disruption of the conserved FGGF motif causes important local effects that affect the structure of the ULBR. - The SH3 domain of human c-Src loses its ability to interact with lipids in presence of a polyproline peptide. - Real-time NMR spectroscopy allowed the study of complex phosphorylation/dephosphorylation processes in USrc mediated by kinases and phosphatases in Xenopus laevis derived cells or cell extracts. - Three different phosphorylation sites present in the Unique domain that were unequivocally assigned to Ser 17, Ser 69 and Ser 75 have been identified in Xenopus laevis, COS-7, MEFs and HeLa cell systems. - It was the first time that phosphorylation of Ser 69 of c-Src was observed in those model systems. - The biological relevance of the ULBR has been firstly demonstrated in Xenopus laevis oocytes in the context of the full-length c-Src. - The functional roles of the described phosphorylation events together with ULBR mutations have been shown in HEK293T cells and in the human colorectal SW620 cancer cell lines in the context of the full-length protein. - A “positional regulation model” has been proposed as a new c-Src selectivity regulation mechanism.
Aquesta tesi, titulada “Caracterització estructural i funcional del domini únic intrínsecament desplegat de c-Src humana”, aborda els aspectes fonamentals de la funció biològica del domini Únic intrínsecament desordenat de c-Src (USrc) que fins ara ha sigut poc estudiat. Hem caracteritzat primer mitjançant tècniques de biologia estructural als efectes de les mutacions de la ULBR (Unique Lipid Binding Region) dirigides al lloc de les interaccions descrites anteriorment amb els lípids i amb el domini SH3. Llavors, hem investigat els esdeveniments de fosforilació que tenen lloc en el domini únic per tècniques de RMN en cèl•lules. Finalment, hem demostrat el paper funcional de USRC en el context de línies de cèl•lules normals i canceroses. En conjunt, els nostres resultats han demostrat que el domini únic de c-Src participa activament en el mecanisme de regulació en què la tirosina quinasa c-Src està implicada. Aquest estudi representa una nova fita essencial en la comprensió de com c-Src pot controlar una àmplia varietat d'esdeveniments de senyalització dins de les cèl•lules. A més, té el potencial d'obrir el camí a la possibilitat de noves aplicacions per al tractament del càncer mitjançant el desenvolupament de nous fàrmacs selectius dirigits residus actius funcionals localització en el domini únic. Aquesta tesi constitueix una "prova de concepte" de com des de la comprensió atomista d'un tema d'interès (enfocament estructural) pot ser possible entendre i successivament demostrar la importància biològica dels fenòmens observats (enfocament funcional).

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22

Ramberger, Evelyn [Verfasser]. "Spatial protein interaction networks of the intrinsically disordered transcription factor CEBPA / Evelyn Ramberger". Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1219065943/34.

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23

Abyzov, Anton. "Nuclear Magnetic Resonance Studies of the Dynamics and Thermodynamics of Intrinsically Disordered Proteins". Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAY026/document.

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Les protéines intrinsèquement désordonnées sont des hétéropolymères très flexibles, impliqués dans des activités cellulaires importantes (transduction du signal, reconnaissance moléculaire, traduction etc.), représentant des cibles potentielles de médicaments contre les maladies neurodégénératives et cancers, et dont les modes dynamiques définissent leur fonction biologique. Même si les états conformationnels qu'elles échantillonnent sont relativement bien connus, ce n'est pas le cas des échelles de temps de la dynamique associée. Dans ce travail nous étudions le comportement conformationnel du domaine C-terminal intrinsèquement désordonné de la nucléoprotéine de virus de Sendai (NTAIL), qui interagit avec le domaine PX de la phosphoprotéine. Des études précédentes montrent que le site d'interaction échantillonne un équilibre entre trois hélices discrètes dans l’état libre, et que l’interaction avec PX passe d’abord par la formation d'un pré-complexe, où l’une des conformation hélicoïdales de NTAIL est stabilisée, puis par sa diffusion sur la surface de PX, et enfin sa rétention sur le site de liaison. Cependant, aucun renseignement n'existe sur les échelles de temps de mouvements de la chaine de NTAIL, qui influencent certainement la cinétique de cette interaction, en particulier sa constante de vitesse d’association. Cette protéine de 124 acides aminés représente aussi un système modèle pertinent contenant à la fois de longs domaines dépliés et des régions de structure résiduelle. La mesure d’un vaste et cohérent ensemble de taux de relaxation à différents champs magnétiques et différentes températures nous a permis de caractériser la dynamique de NTAIL à un niveau de détail sans précèdent. A l’aide d’analyse « model-free » étendu, nous avons montré que les composants rapides de la fonction de corrélation nous informent sur les librations. Le mode dominant se situe à des échelles de temps autour d’une nanoseconde et est lié à l’échantillonnage de l’espace de Ramachandran par le squelette peptidique. Enfin, le composant lent (5-25 ns) nous informe sur les mouvements de segments de la chaine peptidique. La description des mouvements intrinsèques des protéines désordonnées et leurs échelles de temps contribuera à notre compréhension du comportement et des fonctions de ces protéines
Intrinsically disordered proteins (IDPs) are highly flexible heteropolymers, implicated in important cellular activities (signal transduction, molecular recognition, transcription, translation, etc.) and representing potential drug targets against cancer and neurodegenerative diseases, whose dynamic modes define their biological function. Although the conformational states sampled by IDPs are relatively well understood, essentially nothing is known about the associated dynamic timescales. In this study we investigate the conformational behavior of the intrinsically disordered C-terminal domain of the nucleoprotein of Sendai virus (NTAIL), which interacts with the PX domain of the phosphoprotein. The interaction site has been shown to sample an equilibrium of discrete helices in the free state, which forms an encounter complex implicating the stabilization of one of the helical conformers upon interaction with PX, prior to diffusing on the surface of PX and engaging in the actual binding site. However, very little is known about the timescales of chain motions, which surely play a role in the interaction kinetics, in particular in terms of the on-rate of the interaction. This 124 amino acid protein also provides a good model system, containing long unfolded domains with chain-like dynamics and regions with residual structure. The measurement of extensive set of coherent relaxation rates at multiple magnetic fields, multiple temperatures and in three different length constructs of the same IDP has allowed us to characterize the dynamic nature of NTAIL in unprecedented detail. By analyzing the relaxation data using extended model-free approach, we show that fast (≤ 50 ps) components of the correlation function report on librational motions. A dominant mode occurs on timescales around one nanosecond, apparently reporting on backbone sampling within Ramachandran sub-states, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. The ability to delineate intrinsic modes and timescales will improve our understanding of the behavior and function of IDPs

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24

Fu, Biao. "Molecular dynamics simulations of intrinsically disordered proteins with replica-averaged chemical shift restraints". Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709203.

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25

Joshi, Priyanka. "Targeting intrinsically disordered proteins associated with neurodegenerative diseases : a strategy towards drug discovery". Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709234.

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26

Simon, Sharotka M. (Sharotka Maria). "Regulation of E. coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products". Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38994.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.
Includes bibliographical references (p. 202-222).
Products of the umuD gene in E. coli are involved in regulating the timing of error-free DNA repair processes and mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD2 is upregulated early during the SOS response, and a slow post-translational autocleavage process removes the N-terminal 24 amino acids of each UmuD monomer. The remaining C-terminal fragment, UmuD'2, activates the catalytic subunit of the Y family DNA polymerase V for mutagenic TLS. The small proteins UmuD2 and UmuD'2 make a large number of specific protein-protein contacts for their roles in regulation. This report chronicles experiments that indicate that umuD gene products share characteristics with intrinsically disordered proteins, which lack a defined secondary or tertiary structure when purified and many of which have important roles in regulation. Counterintuitively, UmuD2 and UmuD'2 form stable homodimers in vitro at concentrations where little or no a helix or 13 sheet is detectable. High protein concentrations and certain crowding agents can confer more typical secondary structure on UmuD2 and UmuD'2.
(cont.) The binding affinities between UmuD2 and two of its interaction partners are reported; interaction with either of these interaction partners also confers secondary structure on UmuD2. Intrinsic disorder in umuD gene products helps explain how they can make a vast number of specific protein-protein interactions despite their small size, and previous single-cysteine studies of both UmuD2 and UmuD'2 provide insights into the actual structures of intrinsically disordered proteins. A model is presented for how umuD gene products and certain hub proteins, which form highly connected nodes in protein-protein interactomes, can make sequential protein-protein interactions.
by Sharotka M. Simon.
Ph.D.

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27

Beltrandi, Matilde. "Characterization of the intrinsically disordered and multimerization regions of the Henipavirus P proteins". Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4115.

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Le objectif de ma thèse était la caractérisation moléculaire de la P des virus Nipah et Hendra (BL4) du genre Henipavirus. Le génome est encapsidé par la N qui sert de substrat pour la transcription et la réplication. La polymérase est composée par la L e son cofacteur la P. La P est composée d’un domaine N-terminal (PNT) désordonné et un domaine C-terminal (PCT) constitué d’une alternance de régions désordonnés et ordonnés (PMD domaine de multimerization). J'ai étudié PMD, PCT et PNT utilisant le «cross-linking», le CD, le SAXS, la RMN et la modélisation moléculaire. J'ai montré que le PMD du Hendra et Nipah sont un coiled-coil triméreric. La région PCT, est également un trimère en solution. Les protéines P des henipavirus constituent à ce jour le seul exemple de protéines P paramyxovirales ayant une organisation trimérique. En utilisant le SAXS, j'ai obtenu une description de Hendra PNT en tant qu’ensemble conformationnel. J'ai entrepris la caractérisation de la PNT par RMN. J’ai divisée la PNT avec l’approche divide et impera (PNT1,2,3,4). J’ai pu réaliser des expériences permettant l’attribution de PNT1, et j’ai également effectué des mesures de relaxation (R1, R2 et NOE) sur les fragments PNT1, PNT2 et PNT3. Les résultats issus des travaux effectués ont ouvert la voie vers l’obtention d’une description atomistique de la PNT en tant qu'ensemble conformationnel. Ces informations avec les informations structurales que j’ai sur PCT, PMD et XD, devraient conduire à une description atomistique de la P entière en tant qu’ensemble conformationnel. Ces informations structurales détaillées constitueront aussi un socle pour des approches antivirales rationnelles
The objective of my PhD project was the molecular characterization of the P protein from the Nipah and Hendra viruses (BL4) belonging to the Henipavirus genus. The genome is encapsidated by the N that is the substrate for transcription and replication. The polymerase is made up the L and its cofactor the P. The P protein consists of an intrinsically disordered N-terminal domain (PNT), and a C-terminal domain (PCT) made of alternating disordered and ordered domain (PMD or P multimerization domain). I investigated the PMD, PCT and PNT regions, using cross-linking, AUC, CD, SAXS, NMR and molecular modeling. I showed that Hendra and Nipah PMD are a trimeric coiled-coil in solution. The Henipavirus proteins constitute so far the unique examples of a trimeric organization in paramyxoviral P proteins. The PCT is a trimer as well. Using SAXS, I obtained an ensemble description of PNT. To obtain site-specific information that improve SAXS-based models, I undertook the characterization of Hendra PNT by NMR. The latter was divided using the “divide et impera” approach to get four fragments (PNT1,2,3,4). Experiments for the assignment have been performed for PNT1. R1, R2 and NOE were carried out on PNT1,2,3. Altogether the results laid the basis for achieving an atomic-resolution conformational ensemble description of Hendra PNT. This information, combined with structural information that I collected on PCT, PMD and XD, is expected to lead an atomistic ensemble description of the full-length P, which would represent the first, such a description of a paramyxoviral P protein. This detailed structural information will also constitute an asset for rational antiviral approaches

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28

Pinet, Louise. "Structural and functional investigation of the C-terminal intrinsically disordered fragment of ErbB2". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS375/document.

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ErbB2/HER2 est un récepteur tyrosine kinase de la famille d'EGFR (ErbB1) surexprimé dans plus de 20% des cancers du sein et associé à une forme particulièrement agressive de la maladie. Les récepteurs ErbBs sont actifs seulement sous forme de dimères, permettant la phosphorylation de leur queue C-terminale par leur domaine tyrosine kinase. La phosphorylation entraine l'interaction avec des protéines adaptatrices et l'activation de voies de signalisation, Ras/MAPK et PI3K/Akt principalement. Ces voies contrôlent la prolifération, la motilité cellulaire et la résistance à l'apoptose. Contrairement à ErbB1/3/4, ErbB2 dimérise en l'absence de ligand. Comprendre les autres mécanismes de régulation de la phosphorylation de ses tyrosines et de ses interactions est donc particulièrement intéressant.ErbB2 a fait l'objet de nombreuses études structurales et fonctionnelles. Elles ont permis la mise au point de traitements ciblés efficaces mais sujets à l'apparition de résistance, dont l'anticorps Trastuzumab, ciblant sa partie extracellulaire. La queue C-terminale d'ErbB2 (CtErbB2) a été très souvent ignorée dans ces études. Cette partie étant intrinsèquement désordonnée, il a fallu attendre ces dernières années pour que les concepts et les outils permettant de l'étudier émergent.Dans cette thèse, j'ai d'abord effectué la caractérisation structurale et dynamique de CtErbB2. J'ai montré que bien qu'étant dépourvue de toute structure stable, cette région riche en prolines possède plusieurs structures secondaires transitoires et un contact longue-distance participant très probablement à la régulation de ses interactions intra- et inter-moléculaires. Dans une deuxième partie je me suis intéressée à la caractérisation de la protéine adaptatrice Grb2, partenaire essentiel de ErbB2 pour l'activation de la voie des MAP kinases. L'organisation en solution des domaines de cette protéine modulaire dans sa forme libre était jusque là inconnue. J'ai ensuite étudié l'interaction entre Grb2 et CtErbB2, et montré que CtErbB2 interagit non seulement avec le domaine SH2 de Grb2 (par l'intermédiaire d'une phosphotyrosine), mais aussi avec son domaine SH3 N-terminal (grâce à un motif polyproline). Enfin, j'ai mis en place plusieurs stratégies de phosphorylation des tyrosines de CtErbB2, dans le but d'étudier plus largement l'effet des phosphorylations sur l'ensemble de cette région
ErbB2/HER2 is a receptor tyrosine kinase of the EGFR (ErbB1) family overexpressed in 20% of breast cancers and associated to a particularly aggressive form of the disease. ErbB receptors are only active upon dimerization that enables phosphorylation of their C-terminal tail by their tyrosine kinase domain. Phosphorylation then triggers interaction with adaptor proteins and activation of signaling pathways, mainly Ras/MAPK and Akt/PI3K. Those pathways control cell proliferation, motility and resistance to apoptosis. Contrary to ErbB1/3/4, ErbB2 can dimerize without any ligand. Understanding other mechanisms of regulation of its tyrosine phosphorylation and of its interactions is thus particularly interesting.ErbB2 structure and function have been extensively studied. This has led to the development of several FDA-approved targeted drugs, that are effective but to which resistance occurs, amongst which the Trastuzumab antibody that targets ErbB2 extracellular domain. The C-terminal tail of ErbB2 (CtErbB2) has been widely ignored in these studies. Since it is intrinsically disordered, the concepts and tools to study it have only emerged in the last few years.In the present work, I have performed the structural and dynamic study of CtErbB2. I showed that despite its lack of any stable structure, this proline-rich region exhibits several transient secondary structures and a long-range contact that might participate in the regulation of its intra- and inter-molecular interactions. Then, I characterized the adaptor protein Grb2, which is a partner of ErbB2 that is essential for the activation of the MAPK pathway. The solution organization of the domains of this modular protein in its apo-form was unknown so far. I also studied the interaction between Grb2 and CtErbB2, showing that in addition to the known SH2-phosphotyrosine interaction, a polyproline motif of CtErbB2 binds to the N-terminal SH3 domain of Grb2. Finally, I implemented several strategies to phosphorylate CtErbB2 tyrosines, to study more extensively the effect of phosphorylation on the whole tail

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29

Trujillo, Joshua T., Mark A. Beilstein y Rebecca A. Mosher. "The Argonaute-binding platform of NRPE1 evolves through modulation of intrinsically disordered repeats". WILEY-BLACKWELL, 2016. http://hdl.handle.net/10150/622374.

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• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

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30

Protter, David S. W. "Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules". Thesis, University of Colorado at Boulder, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10635323.

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Cells assemble large, non-membrane bound granules of protein and RNA, termed Ri- bonucleoprotein granules (RNP granules), often in response to a wide variety of cellular stresses. This behavior is conserved from yeast to mammals. Some RNP granules ap- pear important in the stress response, while others are important for proper organismal development, and still others for control of RNA degradation and transport. Curiously, proteins found within granules are disproportionately likey to contain Intrinsically Dis- ordered Regions. Here, I show that those disordered regions can often drive higher order assembly in vitro and contribute to granule assembly in vivo. I found that these domains can make it easier for proteins to undergo a process known as Liquid-Liquid Phase Separa- tion in response to changes in ionic strength, wherein the protein of interest self-partitions into a concentrated liquid phase. The droplets that form mimic many of the behaviors of RNP granules in cells, such as recruitment of other IDR-containing proteins, assembly in response to RNA, and rapid exchange of contents with the surrounding medium. I also found that proteins that form these droplets tend to aggregate over time, turning the dynamic droplets into static structures.

Further, I identified several limitations to my in vitro model, most importantly the impairment of IDR-based phase separation in the presence of other proteins or cellular lysates. However, I also helped uncover the synergistic relationship between IDRs and the more well studied protein-protein and protein-RNA interactions that are important for granule assembly. I therefore propose an inclusive model of granule assembly which asserts that a wide variety of types of interactions are important, and that it is the sum-total of these interactions that determines whether or not a granule assembles.

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31

Zhang, Weihong. "Multi-scale simulations of intrinsically disordered proteins and development of enhanced sampling techniques". Diss., Kansas State University, 2014. http://hdl.handle.net/2097/17916.

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Doctor of Philosophy
Department of Biochemistry and Molecular Biophysics
Jianhan Chen
Intrinsically disordered proteins (IDPs) are functional proteins that lack stable tertiary structures under physiological conditions. IDPs are key components of regulatory networks that dictate various aspects of cellular decision-making, and are over-represented in major disease pathways. For example, about 30% of eukaryotic proteins contain intrinsic disordered regions, and over 70% of cancer-associated proteins have been identified as IDPs. The highly heterogeneous nature of IDPs has presented significant challenge for experimental characterization using NMR, X-ray crystallography, or FRET. These challenges represent a unique opportunity for molecular mod- eling to make critical contributions. In this study, computer simulations at multiple scales were utilized to characterize the structural properties of unbound IDPs as well as to obtain a mechanistic understanding of IDP interactions. These studies of IDPs also reveal significant limitations in the current simulation methodology. In particular, successful simulations of biomolecules not only require accurate molecular models, but also depend on the ability to sufficiently sample the com- plex conformational space. By designing a realistic yet computationally tractable coarse-grained protein model, we demonstrated that the popular temperature replica exchange enhanced sampling is ineffective in driving faster reversible folding transitions for proteins. The second original contribution of this dissertation is the development of novel simulation methods for enhanced sampling of protein conformations, specifically, replica exchange with guided-annealing (RE-GA) method and multiscale enhanced sampling (MSES) method. We expect these methods to be highly useful in generating converged conformational ensembles.

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32

Jurneczko, Ewa. "Resolving intrinsically disordered proteins of the cancer genome with ion mobility mass spectrometry". Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/8844.

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For proteins the link between their structure and their function is a central tenet of biology. A common approach to understanding protein function is to ‘solve’ its structure and subsequently probe interactions between the protein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their entire structure fail to fold into a three-dimensional structure and yet they possess function. These so called intrinsically or inherently disordered proteins (IDP’s) or intrinsically disordered regions (IDR’s) constitute up to 40% of all expressed proteins. IDPs which have crucial roles in molecular recognition, assembly, protein modification and entropic chain activities, are often dynamic with respect to both conformation and interaction, so in the course of a protein’s ‘lifespan’ it will sample many configurations and bind to several targets. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. The solvent free environment of a mass spectrometer is ideally suited to the study of intrinsic interactions and how they contribute to structure. Ion mobility mass spectrometry is uniquely able to observe the range of structures an IDP can occupy, and also the effect of selected binding partners on altering this conformational space. This thesis details the technique of ion mobility mass spectrometry and illustrates its use in assessing the relative disorder of p53 protein. The tumour suppressor p53 is at the hub of a plethora of signalling pathways that maintain the integrity of the human genome and regulate the cell cycle. Deregulation of this protein has a great effect on carcinogenesis as mutated p53 can induce an amplified epigenetic instability of tumour cells, facilitating and accelerating the evolution of the tumour. Herein mass spectrometry provides a compelling, detailed insight into the conformational flexibility of the p53 DNA-binding domain. The plasticity of the p53 DNA-binding domain is reflected in the existence of more than one conformation, independent of any conformational changes prompted by binding. The in vacuo conformational phenotypes exhibited by common cancer-associated mutations are determined and the second-site suppressor mutation from loop L1, H115N, is probed whether it could trigger conformational changes in p53 hotspot cancer mutations. The structural basis of the binding promiscuity of p53 protein is investigated; of particular interest is the molecular interaction of the p53 N-terminus with the oncoprotein murine double minute 2, as well as with the antiapoptotic factor B-cell lymphoma-extralarge.

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33

Uluca, Boran [Verfasser]. "Solid-State NMR and DNP-Enhanced NMR Studies on Intrinsically Disordered Proteins / Boran Uluca". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1164763237/34.

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34

Weise, A. "Structural investigations of intrinsically disordered polypeptides : biosynthesis on the ribosome and protein misfolding disease". Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1472901/.

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Intrinsically disordered proteins constitute a significant proportion of our proteome and are emerging targets of modern structural and molecular biology through their involvement in a range of functions crucial for the survival of the cell. Their malfunction and aggregation is known in many cases to lead to devastating pathologies including Parkinson’s disease (α-synuclein, αSyn) and amyloid lateral sclerosis (TAR DNA- binding protein, TDP-43). NMR spectroscopy, is a high resolution structural technique that is especially well suited to provide residue-specific information on flexible and unstructured systems, and which is used to investigate the structural and dynamic aspects of αSyn and TDP-43. TDP-43, is a eukaryotic, 440-residue, multi-domain protein with a predicted disordered C-terminal tail (CTD274-414) that initiates the aggregation of TDP-43 and harbours the disease-associated mutations. This thesis describes the development of an expression and purification strategy and the NMR investigations of the structure and dynamics of the CTD274-414 region both in isolation and as it exists within living cells. Using these methods we have initially obtained the complete protein backbone assignment of the CTD274-414 under 8 M urea conditions, which indicated a mostly disordered conformation, serving now as a valuable template to determine the secondary structure propensity of this domain within E. coli lysate. In addition, the aggregation behaviour of the CTD274-414 was investigated using biophysical techniques, which revealed that while the CTD274-414 aggregates appear to have fibrillar morphology, the overall aggregation properties suggest that CTD274-414 does not form typical β-amyloids. The understanding provided by structural biology over recent years of the PD-associated protein αSyn has enabled detailed insights into the conformational dynamics of several forms of this ’chameleon’ protein. Here, a detailed study of the biosynthesis of αSyn using NMR spectroscopic investigations of ribosome-bound nascent chain complexes (RNCs) is presented. By considering the length-dependent emergence, the effects of charge and also the interaction of αSyn RNCs with the TF chaperone, detailed insights of the interactions arising from the interplay between TF, the ribosome and nascent polypeptide chains were gained. These results were used to create a structural model beginning to reveal the specific manner in which disordered proteins interact with the biosynthesis machinery.

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35

Hibino, Emi. "Physicochemical studies on interaction between intrinsically disordered regions in transcription factors Sp1 and TAF4". 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225524.

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36

Eriksson, Sylvia. "Molecular properties of disordered plant dehydrins : Membrane interaction and function in stress". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-136033.

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Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18).

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

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37

Dibenedetto, Domenica [Verfasser]. "Predicting conformational ensembles of the intrinsically disordered protein alpha-synuclein via molecular simulation / Domenica Dibenedetto". Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/1052303420/34.

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38

Murphy, Sebastien. "Influence of an intrinsically disordered region on peptide binding by the PHear domain of NECAP1". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97257.

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Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrinforms a scaffold around these vesicles and is linked to membranes by clathrin adaptors. The adaptor complexes, AP-2 and AP-1, are key hubs in CCV formation at the plasma membrane and trans-Golgi network (TGN), respectively. They recruit cargo, clathrin and a multitude of accessory proteins. The adaptin, or ear, domains play a critical role in recruiting accessory proteins through binding of specific peptide motifs, such as the FXDXF motif. NECAPs are a family of accessory proteins that enrich in CCVs and associate with AP-2 and AP-1. NECAP1 expression levels are highest in the neuronal cells while NECAP2 is ubiquitously expressed. Structural studies of NECAP1 reveal the fold to be part of the pleckstrin homology (PH) domain superfamily and it mimics AP-2 α-ear binding of FXDXF motifs. Therefore the globular region of NECAP is termed the PHear (PH fold with ear-like function) domain. NECAP1 consists of the PHear domain between amino acids 1-133, and a conserved region that is intrinsically unfolded between 134-178, with the entire unfolded region between amino acids 134-275. This thesis has found using nuclear magnetic resonance (NMR) that the NECAP1 construct 1-178 has a two-fold weaker binding affinity to an FXDXF peptide in comparison to the construct 1-133, with binding constants (KD) of 620 micromolar and 330 micromolar respectively. The decrease in binding affinity is attributed to an interaction between the unfolded region and the PHear domain, with a KD between 4000 and 1000 micromolar.
Les vésicules enduites de clathrine (CCVs pour Clathrin-coated vesicles) sont responsables du transport de protéines entre divers compartiments des systèmes sécréteurs et endocytiques. Les protéines clathrine forment un échafaudage autour de ces vésicules ces dernières sont liées aux membranes par des adaptateurs de clathrine. Les complexes d'adaptateurs, AP-2 et AP-1, sont les points de jonction principaux dans la formation des vésicules enduites de clathrine à la membrane de plasma et au réseau transport-Golgi (TGN), respectivement. Ils recrutent la cargaison, la clathrine et une multitude de protéines accessoires. Les domaines adaptin (aussi appelés domaines oreilles) jouent un rôle critique dans le recrutement des protéines accessoires en se liant à des motifs peptides spécifiques, tels que le motif FXDXF. Les NECAPs sont une famille de protéines accessoires qui se concentrent dans les CCVs et qui s'associent à AP-2 et à AP-1. Les niveaux d'expression NECAP1 sont les plus élevés dans les cellules neuronales tandis que NECAP2 est présente unanimement. Des études structurales de NECAP1 indiquent le pli de la protéine fait d'elle un membre de la superfamille des domaines homologues de pleckstrin (pH). Ces dernières ont comme fonction d'imiter le point d'attache du domaine « α-ear » de AP-2 aux motifs FXDXF. Par conséquent, la région globulaire de NECAP se nomme le domaine PHear (PH fold with ear like function, ou pli PH avec fonction similaire au domaine « ear »).Le domaine PHear de NECAP1 est délimité par les acides aminés 1-133. De plus, la proteine comprend une région conservée qui est intrinsèquement non structurée entre les acides aminés 134 et 178; la totalité de cette région non structuré est comprise entière entre les acides aminés 134 et 275. Cette thèse démontre, en utilisant la résonance magnétique nucléaire (RMN), qu'un segment de NECAP1 composé des acides aminés 1 à 178 a une affinité envers un peptide FXDXF deux fois plus faible que celle d'un segment composé des acides aminés 1 à 133. Les constantes KD étaient de 620 micromolaire et de 330 micromolaire respectivement. La diminution de l'affinité de liaison est attribuée à une interaction entre la région non structurée et le domaine de PHear, avec une constante KD entre 4000 et 1000 micromolaire.

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39

Mei, Yang. "Structural Studies of BECN1, A Key Autophagy Protein, and Intrinsically Disordered Regions in Autophagy Proteins". Diss., North Dakota State University, 2016. https://hdl.handle.net/10365/28030.

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Autophagy, a conserved catabolic process required for cellular homeostasis in eukaryotes, is regulated by many proteins. The central goal of my doctoral research is to investigate conformational flexibility of autophagy proteins, with a special focus on BECN1, a core component of the class III phosphatidylinositol-3 kinase autophagosome nucleation complex that may serve as an autophagy interaction hub. Our rigorous bioinformatics analysis predicts that 57% of 59 key human autophagy proteins contain intrinsically disordered regions (IDRs), which lack stable secondary and tertiary structure. The prevalence of IDRs suggests that IDRs play an important, yet hitherto uninvestigated, role in autophagy. We confirm disorder of selected IDRs via biophysical methods, and use additional bioinformatics tools to predict protein-protein interaction and phosphorylation sites within IDRs, identifying potential biological functions. We experimentally investigate four distinct BECN1 domains: (i) The IDR, which includes a functional BCL2 homology 3 domain (BH3D) that binds BCL2 proteins, undergoing a binding-associated disorder-to-helix transition and enabling BCL2s to inhibit autophagy. (ii) The flexible helical domain (FHD) which has an unstructured N-terminal half and structured Cterminal half forming a 2.5-turn helix in our 2.0 ? X-ray crystal structure. Our molecular dynamics simulations and circular dichroism spectroscopy analyses indicate the FHD transiently samples more helical conformations and likely undergoes a binding-associated disorder-to-helix transition. We also show that the FHD bears conserved residues critical for AMBRA1 interaction and for starvation-induced autophagy. (iii) A coiled-coil domain (CCD) which forms an antiparallel homodimer in our 1.46 ? X-ray crystal structure. We have also built a atomistic model of an optimally packed, parallel BECN1:ATG14 CCD heterodimer that agrees with our experimental SAXS data. Further, we show that BECN1:ATG14 heterodimer interface residues identified from this model are important for heterodimer formation and starvation-induced autophagy. (iv) A ?-? repeated autophagy-specific domain which bears invariant residues that we show are important for starvation-induced autophagy. Thus, we demonstrate that conformational flexibility is a key BECN1 feature. Lastly, we show that multi-domain BECN1 constructs have extended conformations with no intra-domain interactions that impact structure of other domains, suggesting that BECN1 structure and conformational flexibility enable its function as an autophagy interaction hub.

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40

McDowell, Chester Dale. "Potential heterogeneity in p53/S100B(ββ) complex". Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/13845.

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Master of Science
Department of Biochemistry
Jianhan Chen
Paul E. Smith
Intrinsically disordered proteins have been shown to be important in many physiological processes, including cell signaling, translation, and transcription. They are also associated with cancer, and neurodegenerative diseases. The tumor suppressor p53 contains several disordered regions, including the C-terminal negative regulatory domain (NRD). In cancer the function of p53 has been shown to be repressed by S100B(ββ) binding to p53-NRD. Binding of S100B(ββ) blocks acetylation and phosphorylation sites in the p53-NRD, which leads to tetramer dissociation and prevents p53 activation. NMR studies have shown that p53-NRD binds S100B(ββ) in a stable α-helix conformation. Interestingly, despite the well-converged and apparent rigid nature of the NMR structure ensemble, a majority of intermolecular NOEs used to calculate the NMR ensemble are very weak (≥6 Å). The final NMR structures also contains unsatisfied buried charged residues at the binding interface. It’s plausible that the p53-S100B(ββ) complex is more dynamic than previously believed. The goal of the study is to determine the potential conformational heterogeneity in p53-S100B(ββ) complex using molecular modeling. For this, five diverse structures were selected from the 40-member NMR ensemble. For each initial conformation, we performed 100 ns molecular dynamic simulations in explicit solvent to explore the structure and dynamics of the p53-NRD in complex with S100B(ββ). Several analytical tools were used to characterize the p53-NRD conformation, including root-mean squared deviation (RMSD), root-mean squared fluctuation (RMSF), and residue helicity. The accuracy of the simulations was mainly assessed by comparing with experimental NOEs. The results show that, even though the ensemble is heterogeneous it satisfies 82% of the experimental NOEs. Clustering analysis further suggests that many conformational sub-states coexist for this complex, and individual clusters appear to satisfy only subsets of NOE distances. Importantly, the buried surface analysis demonstrates that the heterogeneous ensemble generated from MD provides similar shielding of key residues, which include post-translational modification residues needed for p53 activation. This study also demonstrates that atomistic simulations can provide important insights into structure and dynamics of IDPs for understanding their biological function.

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41

Burkart-Solyom, Zsofia. "NMR methods for intrinsically disordered proteins : application to studies of NS5A protein of hepatitis C virus". Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENY077/document.

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Les protéines intrinsèquement désordonnées sont caractérisées par un manque de structure 3D stable et sont biologiquements actives dans cet état. La spectroscopie RMN est la méthode de choix pour leurs études à une résolution atomiques, car la cristallographie aux rayons X ne permet pas leur étude en raison de leur caractère hautement dynamique.Cependant, l'étude par spectroscopie RMN de ces protéines est difficiles à cause du grand nombre de recouvrement entre les signaux dans le spectre résultant de l'absence d'un réseau de liaison hydrogène qui pourrait stabiliser la structure et permettre d'obtenir une dispersion des signaux plus élevé. Un autre problème est la sensibilité expérimentale car souvent le temps de mesure est limité en raison de leur prédisposition à la dégradation protéolytique. Dans la première partie de cette thèse les protéines intrinsèquement désordonnées sont introduites. La deuxième partie porte sur la spectroscopie RMN des protéines intrinsèquement désordonnées, des expériences RMN de type BEST-TROSY sont présentées et sont montrées comme étant bien adapté pour l'étude de protéines intrinsèquement désordonnées, en particulier pour celle avec une grande étendue de structure résiduelle. Des expériences 3D BEST-TROSY sont présentées pour leur attribution, une version proline-éditée permet d'aider à l'identification de ce type d'acide aminé et enfin l'expérience HETex-BEST-TROSY qui permet une mesure rapide des taux de change de solvants. Dans la troisième partie de cette thèse ces expériences RMN sont appliquées pour l'étude de la région intrinsèquement désordonnés (domaines 2 et 3) de la protéine NS5A du virus de l'hépatite C (VHC). La structure secondaire résiduel présente dans le fragment de la protéine est analysée. La comparaison des données RMN sur trois constructions de la protéine de différentes longueurs ainsi que les données de SAXS permettent l'identification des interactions transitoires à longue portée entre les différentes régions de cette protéine. En outre, les modes de liaison de ce fragment de protéine à Bin1 domaine SH3 sont analysés. Enfin, les résultats préliminaires obtenus sur l'étude de la phosphorylation de NS5A du VHC par certaines kinases, qui ont été montrées comme biologiquement pertinents, sont présentés
Intrinsically disordered proteins are characterized by a lack of a stable, 3D structure and fulfill their biological role as such. NMR spectroscopy is the method of choice for their atomic resolution studies, as X-ray crystallography is not amenable to them due to their highly dynamic character.However, NMR spectroscopic studies of these proteins are challenging, because of the high extent of signal overlap in the spectra, resulting from the absence of a hydrogen-bonding network that would lead to structuring and higher signal dispersion. A further problem is experimental sensitivity as often measurement time is limited due to their predisposition for proteolytic degradation. In the fist part of this thesis intrinsically disordered proteins are introduced. The second part focuses on NMR spectroscopy of IDPs, BEST-TROSY-type NMR methods are presented and are shown to be well suited for large IDPs, especially for those with high extent of residual structure. 3D BEST-TROSY experiments are presented for assignment, a proline-edited version for aiding amino acid-type identification, and the HETex-BEST-TROSY experiment that allows rapid measurement of solvent exchange rates. In the third part of this thesis NMR methods are applied for study of the entire intrinsically disordered region (domains 2 and 3) of NS5A protein of hepatitis C virus. The residual secondary structure in this protein fragment is analyzed. Comparison of NMR data on three protein constructs of different lengths together with SAXS data allows identification of transient long range interactions between different regions of this protein. Furthermore, the binding modes of this protein fragment to Bin1 SH3 domain are analyzed. Finally, the preliminary results obtained on investigation of phosphorylation of NS5A of HCV by certain kinases, reported to be biologically relevant, are presented

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42

Myers, Janette Bernadette. "Application of Single Particle Electron Microscopy to Native Lens Gap Junctions and Intrinsically Disordered Signaling Complexes". PDXScholar, 2019. https://pdxscholar.library.pdx.edu/open_access_etds/5008.

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Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part to the lack of high-resolution (atomic-level) structural knowledge on this class of proteins. Prior to this work, the only high-resolution information available on gap junction structure came from a single connexin isoform, connexin-26 (cx26). CryoEM has recently transformed from a low-resolution technique into one capable of rivaling the atomic-level resolutions achieved by x-ray crystallography -- but without the necessity for crystal formation, which has hindered progress towards understanding many classes of proteins (ie, membrane proteins, intrinsically disordered cell signaling complexes and other structurally dynamic systems). For my thesis research, I applied novel methods in single particle electron cryo-microscopy (CryoEM) to study a class of membrane proteins called gap junctions isolated from native lens tissue, as well as two signaling complexes not amenable to other structural techniques. I determined the structure of the lens gap junction, which contains connexin-46 (cx46) and connexin-50 (cx50), to a resolution of 3.4 Å and generated atomic models for both connexin isoforms. Structural analysis paired with molecular dynamics gave insight into energetic features of these channels that determine their isoform-specific conductance and selectivity to electrically charged ions. The cx46/50 gating domain was found to be stabilized by hydrophobic anchors, and also seems to adopt a more stable open state than found in cx26. Genetic mutations associated with congenital cataract formation were found to map to hot-spots of conserved structural and functional importance, rationalizing their disease-causing effects. As part of collaborative efforts, I used methods in single particle EM to characterize two separate signaling complexes that had proven difficult to study with x-ray crystallography and NMR spectroscopy. One system, Ca2+/Calmodulin Kinase II (CaMKII), is a signaling complex in the brain involved in memory formation. Characterization of the CaMKII complex by single particle EM revealed an extended state, which was also shown to be prevalent in cells -- giving more depth to our understanding of how this signaling molecule functions. The second collaboration characterized the multimeric binding sites of the hub protein LC8, which interacts with the disordered region of a transcription factor (ASCIZ). This provided support for a novel model of transcription regulation, wherein LC8 fine-tunes its own transcription levels through multi-valent binding to the disordered region of its own regulatory transcription factor.

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43

Dahal, Liza. "Functional relevance of protein disorder : why is disorder favourable?" Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/283008.

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For half a century, the central tenet of protein science has been grounded on the idea that the three-dimensional structure of a protein underlies its function. However, increasing evidence of natively unstructured but functional proteins is accumulating. Termed as intrinsically disordered proteins (IDPs), they populate a number of different conformations in isolation. Interestingly, as part of their function, some IDPs become fully or partly structured upon interaction with their binding partners. This process, known as coupled folding and binding raises the question what comes first - folding of the IDP or binding to its partner protein followed by folding. This thesis focuses on understanding the role of disorder in protein- protein interactions using biophysical characterization. Over-representation of IDPs in complex network and signalling pathways emphasizes the importance of disorder. Conformational flexibility in IDPs facilitates post-translational modifications, which provides a neat way to modulate the residual structure. This can alter affinity of IDPs to their partners and it is speculated that bound like structures of IDPs speed association. The impact of phosphorylation was explored in the KID/KIX system: phosphorylation modulates only the dissociation kinetics increasing the lifetime of the bound complex, which may be important in signalling processes. Further, phi-value analysis applied to investigate the mechanism of interaction reveals that non-native interactions play a key role in this reaction, before the IDP consolidates its final structure in the bound complex. Promiscuous interaction of IDPs with their partners often results in complexes with differing affinities. Members of BCL-2 family were explored, and the results indicate that IDPs bind to the same partner protein with marginal variation in the association rates, but significant differences in dissociation rates are observed. Thus, it seems that in such homologous but competing network of proteins, disorder facilitates complexes with differing affinities by modulating dissociation rate, again altering the lifetime of the bound complex. The work presented here demonstrates that disorder plays a role in altering complex lifetimes. Perhaps being disordered permits a level of plasticity to IDPs to adapt the rates at which they bind/unbind to many target proteins. This may be why disorder is conserved and abundant in proteins involved in intricate signalling networks.

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44

Wilkinson, Nina. "An analysis of intrinsically disordered proteins using hidden Markov models and experimental design of stochastic kinetic models". Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2846.

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An intrinsically disordered protein (IDP) is a protein without a stable secondary or tertiary structure and just over one third of human proteins can be described as IDPs. There has been shown to be a link between neurodegenerative diseases, cancer and protein misfolding, with many of these misfolded proteins being intrinsically disordered. These IDPs may be cytotoxic by interacting and contributing to the aggregation process, which is why cells need to regulate these proteins carefully. Research has shown that hydrophobicity and charge may be important in determining if the amino acid sequence has unstructured areas. We study the sequence structure by rst recoding amino acid sequences according to their hydrophobicity and charge and then tting a hidden Markov model using Markov chain Monte Carlo methods to analyse the sequence structure and use a power posterior analysis to determine the number of distinct transition structures. The results show there to be distinct segment types within the amino acid sequences of the FET proteins which may have biological importance. The location of these segments can be used to guide laboratory work which tests the biological signi cance of these segment types within cells. One particular segment found in the FET proteins has been linked to oncogenic fusion proteins and experimental analysis has shown a link between this segment and oncogenic activity. When conducting an experiment, an experimenter needs to determine when and under what conditions they should take measurements. Often the choice of optimal design is made with respect to some statistical criteria. The aim of this work is to determine, for a stochastic kinetic model, the optimal location of the timepoints at which observations are taken. Commonly the statistical criteria involves maximising a utility function over the prior predictive distribution of possible experimental outcomes. Current methodologies for experimental design for models with intractable likelihoods are very computationally expensive as, within the iterative search for the optimal design, the calculation of the utility function requires the determination of the parameter posterior distribution at each iteration. We show how to use delta methods and a Gaussian process as an emulator for the utility to reduce the computational cost and illustrate their application for the simple death process and the Lotka{Volterra model.

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45

Yu, Lei. "Molecular Dynamics of Folded and Disordered Polypeptides in Comparison with Nuclear Magnetic Resonance Measurement". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1521650422218055.

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46

Cattani, Julia Teresa [Verfasser]. "Spectroscopic Investigation of the Intrinsically Disordered Protein Alpha-Synuclein in vitro and in the Cell / Julia Teresa Cattani". Konstanz : KOPS Universität Konstanz, 2017. http://d-nb.info/1202012825/34.

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47

Robotta, Marta [Verfasser]. "The Intrinsically Disordered Protein Alpha-Synuclein and its Membrane Interactions Studied by Electron Paramagnetic Resonance Spectroscopy / Marta Robotta". Konstanz : Bibliothek der Universität Konstanz, 2014. http://d-nb.info/1148942041/34.

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48

Hill, Stephanie. "Folding and association of spectrin tetramer domains : a study of intrinsically disordered proteins from a protein folding perspective". Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648579.

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49

Bomblies, Rainer [Verfasser], Martin [Akademischer Betreuer] [Gutachter] Zacharias y Iris [Gutachter] Antes. "Structure and Dynamics of Intrinsically Disordered Proteins upon Phosphorylation / Rainer Bomblies ; Gutachter: Martin Zacharias, Iris Antes ; Betreuer: Martin Zacharias". München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1133261981/34.

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50

Fisher, Charles. "Statistical Characterization of Protein Ensembles". Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10223.

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Conformational ensembles are models of proteins that capture variations in conformation that result from thermal fluctuations. Ensemble based models are important tools for studying Intrinsically Disordered Proteins (IDPs), which adopt a heterogeneous set of conformations in solution. In order to construct an ensemble that provides an accurate model for a protein, one must identify a set of conformations, and their relative stabilities, that agree with experimental data. Inferring the characteristics of an ensemble for an IDP is a problem plagued by degeneracy; that is, one can typically construct many different ensembles that agree with any given set of experimental measurements. In light of this problem, this thesis will introduce three tools for characterizing ensembles: (1) an algorithm for modeling ensembles that provides estimates for the uncertainty in the resulting model, (2) a fast algorithm for constructing ensembles for large or complex IDPs and (3) a measure of the degree of disorder in an ensemble. Our hypothesis is that a protein can be accurately modeled as an ensemble only when the degeneracy of the model is appropriately accounted for. We demonstrate these methods by constructing ensembles for K18 tau protein, \(\alpha\)-synuclein and amyloid beta - IDPs that are implicated in the pathogenesis of Alzheimer's and Parkinson's diseases.

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