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1
Gill, Katrina Louise. "Protein-protein interactions in membrane proteins". Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400016.
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2
Stylianou, Julianna. "Protein-protein interaction of HSV-1 tegument proteins". Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24663.
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Herpes simplex virus type 1 virions contain a proteinaceous layer between the nucleocapsid and the virus envelope termed the tegument. The mechanism underlying tegumentation remains largely undefined for all herpesviruses, as does the role of many tegument proteins in virus replication. The networks of protein interactions involved in virus assembly have been largely explored and although large-scale studies have been carried out using yeast two hybrid analyses of herpesvirus protein interactions, few of the identified networks have been validated in infected cells. Here, the molecular interactions that occur between the major tegument proteins VP22, VP16 and VP13/14 and a range of glycoproteins and tegument proteins were defined in detail. Two alternative studies were performed from infected cells, however one based on the purification of GFP-tagged proteins and their protein partners proved more successful. These studies validated previous findings and also identified VP13/14, UL21, UL16 and vhs as novel binding partners of VP22, and VP22, UL21, UL16 and vhs as novel binding partners of VP13/14. Thus, these results have led to the identification of two discrete tegument protein complexes in the infected cell: VP22-VP16-VP13/14-vhs and VP22-VP13/14-UL21-UL16. To investigate the nature of the VP22-VP16-VP13/14-vhs complex in more detail, a number of techniques were used and showed that VP22 and VP13/14 both bind directly to the C-terminus of VP16, but were unable to interact with each other. As anticipated from other studies on transfected cell extracts, vhs was shown to be incorporated into this complex by virtue of its direct binding to VP16 during infection, and did not have the capacity to interact directly with VP22. This work has established a defined network of protein-protein interactions encompassing over one third of tegument proteins, and will improve our understanding of the wider protein interaction networks that lead to the assembly of the herpesvirus tegument.
3
Folkman, Lukas. "Predicting Stability and Functional Changes Induced by Protein Mutations with a Machine Learning Approach". Thesis, Griffith University, 2015. http://hdl.handle.net/10072/367589.
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Proteins form a group of one of the most vital macromolecules in living organisms. Yet, even a single mutation in a protein sequence may result in significant changes in protein stability, structure, and thus in protein function as well. Therefore, reliable prediction of stability changes induced by protein mutations is an important aspect of computational protein design, which can aid novel medical and technological discoveries. Also, many mutations have a functional impact which may lead to a disease. Therefore, a key component of personalised medicine is to fully annotate human genetic variations among different individuals. Obviously, it would be infeasible to examine the impact of each possible variant experimentally. Instead, computational methods are needed for a quick and large-scale annotation of genetic variants.
In this thesis, we proposed machine learning methods for predicting stability changes induced by single amino acid substitutions and for detecting disease-causing frameshifting indels (genetic variants caused by short insertions and deletions in the DNA sequence) and nonsense mutations (single nucleotide variants which truncate the protein sequence). The proposed methods can predict the effects of these mutations without the knowledge of the protein structure, which make them applicable universally to all proteins encoded in the human genome.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Information and Communication Technology
Science, Environment, Engineering and Technology
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4
Li, Wei. "Protein-protein interaction specificity of immunity proteins for DNase colicins". Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302033.
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5
Wang, Chu. "Improved conformational sampling for protein-protein docking /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9194.
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6
Sarkar, Mohosin M. "Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility". The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.
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7
Xu, Ping. "Sensing and analyzing unfolded protein response during heterologous protein production :". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 205 p, 2008. http://proquest.umi.com/pqdweb?did=1555621341&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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8
Flöck, Dagmar. "Protein-protein docking and Brownian dynamics simulation of electron transfer proteins". [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969418736.
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9
Marri, Lucia <1977>. "CP12: Intrinsically Unstructured Proteins regulating photosynthetic enzymes through protein-protein interactions". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/423/1/LMarri-BiolFunzSistCellMol-XIX.pdf.
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10
Marri, Lucia <1977>. "CP12: Intrinsically Unstructured Proteins regulating photosynthetic enzymes through protein-protein interactions". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/423/.
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11
Tse, Muk-hei. "Investigations on recombinant Arabidopsis acyl-coenzyme A binding protein 1". View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36427664.
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12
Wang, Hua. "Control of protein-surface, protein-protein, and cell-matrix interactions for biomaterials as tissue engineering scaffolds /". Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9894.
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13
Prigge, Justin Robert. "Identification and characterization of novel protein-protein interactions with the basal transcription factor, TATA-binding protein". Diss., Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/prigge/PriggeJ0506.pdf.
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14
Pateman, Cassandra Sophie Catherine. "RGS proteins and G protein signalling". Thesis, University of Warwick, 2002. http://wrap.warwick.ac.uk/2367/.
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The work within this thesis is concerned with the creation of a temperature-sensitive Schizosaccharomyces pombe marker protein, and the regulation of the pheromone communication system of Sz. pombe reporter strains by RGS proteins. There are a limited number of marker proteins available for use in the genetic manipulation of Sz. pombe, and the generation of a temperature-sensitive Ura4p was envisaged to expand the scope of carrying out sequential gene disruptions in the fission yeast. PCR-based mutagenesis was used to introduce mutations in the ura4 cassette, and a leucine to proline mutation identified at residue 261 in the ura4 open reading frame conferred a temperature-sensitive requirement for uracil. To demonstrate the use of the Ura4sp marker in gene disruption, the Sz. pombe irpl gene was disrupted with the ura4u cassette, and subsequently, the prkl gene was disrupted with the wild-type ura4 cassette. RGS proteins are a recently discovered family of proteins that negatively regulate G protein-coupled signalling pathways. This thesis describes the ability of mammalian RGS proteins to regulate the pheromone communication system of Sz. pombe reporter strains. Human RGS 1 and human RGS4 displayed the greatest ability to negatively regulate the Sz. pombe pheromone signalling pathway when expressed from multicopy expression vectors. Human RGS2, human RGS3, human RGS9-2 and murine RGS2 displayed lesser, varying abilities. Expression of human RGS 1 from single copy reduced signalling at low pheromone concentrations. Expression of human RGS4 from single copy was incapable of reducing pheromone-independent and pheromone-dependent signalling. This thesis also describes the search for gain-of-function RGS proteins. Two potential gain-of-function szRgslp mutants were previously identified, and these mutants were recreated. The two mutations identified (histidine to arginine at szRgslp residue 171 and valine to isoleucine at szRgslp residue 305) conferred gain-of-function szRgslp phenotypes in an sxa2:: ura4 reporter strain. Hydroxylamine treatment of the human RGS4 open reading frame resulted in the identification of a potential gain-of-function RGS4 mutant. The lysine to arginine mutation at huRGS4p residue 20 conferred a gain-of-function huRGS4p phenotype in an sxa2:: ura4 reporter strain.
15
Ratanji, Kirsty. "Investigating the immunogenicity of therapeutic proteins : protein aggregation and host cell protein impurities". Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-immunogenicity-of-therapeutic-proteins-protein-aggregation-and-host-cell-protein-impurities(fda43dd8-2c1e-492b-abd9-e010774d2219).html.
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The development of anti-drug antibodies (ADA) against therapeutic proteins can impact upon drug safety and efficacy. This is a major challenge in the development of biotherapeutics. Various factors have the potential to contribute to protein immunogenicity and the production of ADA. Protein aggregation is one of these factors, though the mechanisms underlying aggregate immunogenicity are poorly understood. In this thesis the effect of protein aggregation on immunogenicity has been investigated. The thermal and/or mechanical stresses required in order to achieve subvisible aggregates of three test proteins were determined. Stressed preparations of proteins were characterised using a suite of biophysical techniques, including dynamic light scattering and circular dichroism. The immunogenic potential of subvisible aggregates of a humanised single chain variable fragment (scFv) and ovalbumin (OVA) was studied following intraperitoneal exposure in BALB/c strain mice. Monomeric proteins induced a T helper (Th) 2 dominant immune response, but when aggregated, the responses gained a Th1 phenotype, with a significant increase in the antigen-specific IgG2a antibody response. Cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were also consistent with aggregated preparations of OVA inducing a Th1-type response. Host cell protein (HCP) impurities can also contribute to immunogenicity. Mass spectrometry analysis of an scFv preparation identified the presence of the Escherichia coli (E.coli) heat shock protein DnaK, amongst other HCP, as an impurity. Protein preparations free from DnaK were spiked with recombinant E.coli DnaK to mimic the HCP impurity. The effect of DnaK on the immunogenicity of aggregated and monomeric scFv preparations was then investigated. BALB/c mice were immunised with monomeric and aggregated preparations, with and without E.coli DnaK at 0.1% by mass. Aggregation alone resulted in an enhanced IgG2a antibody response, and the presence of DnaK increased this further. Comparable investigations were also conducted using mouse albumin; here an increase in immunogenicity was observed with protein aggregation, and the presence of DnaK was found to increase the IgG2a response. Collectively, the evidence presented in this thesis shows that aggregation can impact upon the magnitude and character of induced immune responses, and that subvisible aggregation promotes a Th1 immune skewing. Additionally, E.coli HCP DnaK enhances protein aggregate immunogenicity, which indicates that heat shock proteins, as a class of HCP, could have an adjuvant-like effect on biotherapeutic aggregates.
16
Almeida, T. B. "Identification and optimisation of ligands to target protein-protein interactions : EB1-SxIP proteins". Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004877/.
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End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule growing ends which has a fundamental role in microtubule polymerisation. EB1 regulates the microtubule dynamic behaviour, through protein recruitment, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved SxIP motif within an intrinsically disordered region enriched with basic, serine and proline residues. Crystal structure of EB1 in complex with a peptide containing the SxIP motif demonstrated that the isoleucine-proline dipeptide is bound into a well‐defined cavity of EB1 that may be suitable for small molecule targeting. The research described herein reports the use of a multidisciplinary approach for the discovery of the first small molecule scaffold to target the EB1 recruiting domain. This approach included virtual screening (structure and ligand based design) and multiparameter compound selection. Solution NMR structures of the C-terminal domain of EB1 in the free form and in complex with the small molecule are also reported. A key finding from these structures is that the hydrophobic binding pocket reported to be essential for recruiting SxIP proteins is not pre-formed but highly dynamic in solution. This brings new insights to the protein recruitment mechanism regulated by EB1 and for the identification of new small molecule inhibitors for the EB1-SxIP protein interactions. The interaction of short length peptides containing the SxIP motif with EB1 was characterised through the use of solution NMR and ITC methods. The contributions for the binding of the SxIP motif and neighbouring residues to EB1 were quantified in terms of binding energy. A structural model shows that the binding pocket of EB1 is largely extended when in complex. This research describes not only the first chemical scaffold that targets EB1, it details important structural features of the interaction of this protein with SxIP containing peptides. This structural information provides fundamental understanding of this interaction that can be exploited in the future to discover higher affinity ligands.
17
Diaz, Manisha Regina. "Use of bionanotechnology to decipher the patterns of assemblage and interactions of multi-protein complexes". Bowling Green, Ohio : Bowling Green State University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=bgsu1250955267.
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18
Simons, Kim T. "Deciphering the protein folding code : ab initio prediction of protein structure /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/9234.
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19
Kundra, Rishika. "Homeostasis of metastable proteins in Alzheimer's disease". Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/268485.
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Alzheimer’s disease (AD) is the most common cause of dementia, affecting almost 40 million people worldwide, and it is predicted that this number will rise to nearly 150 million by 2050. It results not only in enormous distress for affected individuals and carers but also a substantial economic burden on society. Although more than 100 years have passed since its discovery, no cure for AD exists, despite enormous efforts in basic and clinical research over the past few decades, due to limited understanding of its underlying mechanisms. Neurodegenerative disorders, of which AD is an example, are highly complex disorders characterized by extensive neuronal dysfunction associated with the misfolding and aggregation of a specific set of proteins, including amyloid plaques and neurofibrillary tangles in AD. One promising avenue for progress in the field is to improve our understanding of the mechanisms by which cellular dysfunction arises from the initial protein aggregation events. The studies described in the thesis are based on the recent finding that a large number of proteins are inherently supersaturated, being expressed at concentrations higher than their solubilities, and constituting a metastable subproteome potentially susceptible to aggregation. These studies illustrate the dependence of aggregation prone metastable proteins on the cellular degradation machineries. They also study the role of metastable proteins and their homeostasis complement in the vulnerability of various body and brain tissues to protein aggregation diseases. Using extensive sequencing data and network based systems biology approaches, they elucidate how fundamental physicochemical properties of an individual or group of proteins relate to their biological function or dysfunction.
20
Watt, Stephen J. "Use of electrospray ionization mass spectrometry to study protein conformation and protein-protein interactions". Access electronically, 2005. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20060516.114814/index.html.
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Thesis (Ph.D.)--University of Wollongong, 2005.Typescript. EMBARGOED-this thesis is subject to a six months embargo (07/09/06) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 159-194.
21
Nguyen, Giang Huong. "A functional analysis of the human LPA₁G protein coupled receptor". Thesis, Available online, Georgia Institute of Technology, 2004:, 2004. http://etd.gatech.edu/theses/available/etd-06072004-131304/unrestricted/nguyen%5Fgiang%5Fh%5F200405%5Fms.pdf.
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22
Jüttemann, Thomas. "Adding 3D-structural context to protein-protein interaction data from high-throughput experiments". Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5666.
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In the past decade, automatisation has led to an immense increase of data in biology. Next generation sequencing techniques will produce a vast amount of sequences across all species in the coming years. In many cases, identifying the function and biological role of a protein from its sequence can be a complicated and time-intensive task. The identification of a protein's interaction partners is a tremendous help for understanding the biological context in which it is involved. In order to fully characterise a protein-protein interaction (PPIs), it is necessary to know the three-dimensional structure of the interacting partners. Despite optimisation efforts from projects such as the Protein Structure Initivative, determining the structure of a protein through crystallography remains a time- and cost-intensive procedure. The primary aim of the research described in this dissertation was to produce a World Wide Web resource that facilitates visual exploration and validation (or questioning) of data derived from functional genomics experiments, by building upon existing structural information about direct physical PPIs. Secondary aims were (i) to demonstrate the utility of the new resource, and (ii) its application in biological research. We created a database that emphasises specifically the intersection between the PPIs-results emerging from the structural biology and functional genomics communities. The BISC database holds BInary SubComplexes and Modellable Interactions in current functional genomics databases (BICS-MI). It is publicly available at hyyp://bisc.cse.ucsc.edu. BISC is divided in three sections that deliver three types of information of interest to users seeking to investigate or browse PPIs. The template section (BISCHom and BISCHet) is devoted to those PPIs that are characterised in structural detail, i.e. binary SCs extracted from experimentally determined three-dimensional structures. BISCHom and BISCHet contain the homodimeric (13,583 records) and heterodimeric (5612 records) portions of these, respectively. Besides interactive, embedded Jmol displays emphasising the interface, standard information and links are provided, e.g. sequence information and SPOP classification for both partners, interface size and energy scores (PISA). An automated launch of the MolSurfer program enables the user to investigate electrostatic and hydrophobic correlation between the partners, at the inter-molecular interface. The modellable interactions section (BISC0MI) identifies potentially modellable interactions in three major functional genomics interaction databases (BioGRID), IntAct, HPRD). To create BISC-MI all PPIs that are amenable to automated homology modelling based on conservative similarity cut-offs and whose partner protein sequences have recrods in the UniProt database, have been extracted. The modellable interaction services (BISC-MI Services) section offers, upon user request, modelled SC-structures for any PPIs in BISC-MI. This is enabled through an untomated template-based (homology) modelling protocol using the popular MODELLER program. First, a multiple sequence alignment (MSA) is generated using MUSCLE, between the target and homologous proteins collected from UniProt (only reviewed proteins from organisms whose genome has been completely sequenced are included to find putative orthologs). Then a sequence-to-profile alignment is generated to integrate the template structure in the MSA. All models are produced upon user request to ensure that the most recent sequence data for the MSAs are used. Models generated through this protocol are expected to be more accurate generally than models offered by other automated resources that rely on pairwise alignments, e.g. ModBase. Two small studies were carried out to demonstrate the usability and utility of BISC in biological research. (1) Interaction data in functional genomics databases often suffers from insufficient experimental and reporting standards. For example, multiple protein complexes are typically recorded as an inferred set of binary interactions. Using the 20S core particle of the yeast proteasome as an example, we demonstrate how the BISC Web resource can be used as a starting point for further investigation of such inferred interactions. (2) Malaria, a mosquito-borne disease, affects 3500-500 million people worldwide. Still very little is known about the malarial parasites' genes and their protein functions. For Plasmodium falciparum, the most lethal among the malaria parasites, only one experimentally derived medium scale PPIs set is available. The validity of this set has been doubted in the the malarial biologist community. We modelled and investigated eleven binary interactions from this set using the BISC modelling pipeline. Alongside we compared the BISC models of the individual partners to those obtained from ModBase.
23
Gao, Wei, e 高威. "Characterization of protein interactors of Arabidopsis acyl-coenzymea-binding protein 2". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43223837.
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24
Joachimiak, Lukasz A. "In silico evolution of protein-protein interactions : from altered specificities to de novo complexes /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9211.
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25
Guney, Tacettin Dogacan. "Prediction Of Protein-protein Interactions From Sequence Using Evolutionary Relations Of Proteins And Species". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12611058/index.pdf.
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Prediction of protein-protein interactions is an important part in understanding the biological processes in a living cell. There are completely sequenced organisms that do not yet have experimentally verified protein-protein interaction networks. For such organisms, we can not generally use a supervised method, where a portion of the protein-protein interaction network is used as training set. Furthermore, for newly-sequenced organisms, many other data sources, such as gene expression data and gene ontology annotations, that are used to identify protein-protein interaction networks may not be available. In this thesis work, our aim is to identify and cluster likely protein-protein interaction pairs using only sequence of proteins and evolutionary information. We use a protein&rsquos phylogenetic profile because the co-evolutionary pressure hypothesis suggests that proteins with similar phylogenetic profiles are likely to interact. We also divide phylogenetic profile into smaller profiles based on the evolutionary lines. These divided profiles are then used to score the similarity between all possible protein pairs. Since not all profile groups have the same number of elements, it is a difficult task to assess the similarity between such pairs. We show that many commonly used measures do not work well and that the end result greatly depends on the type of the similarity measure used. We also introduce a novel similarity measure. The resulting dense putative interaction network contains many false-positive interactions, therefore we apply the Markov Clustering algorithm to cluster the protein-protein interaction network and filter out the weaker edges. The end result is a set of clusters where proteins within the clusters are likely to be functionally linked and to interact. While this method does not perform as well as supervised methods, it has the advantage of not requiring a training set and being able to work only using sequence data and evolutionary information. So it can be used as a first step in identifying protein-protein interactions in newly-sequenced organisms.
26
Hetti, Arachchilage Madara Dilhani. "Coevolution of epitopes in HIV-1 pre-integration complex proteins: protein-protein interaction insights". Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1530646538935895.
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27
Zwart, Lizahn. "Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4". Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted
28
Hon, Jiří. "Vyhledávání příbuzných proteinů s modifikovanou funkcí". Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2015. http://www.nusl.cz/ntk/nusl-234914.
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Protein engineering is a young dynamic discipline with great amount of potential practical applications. However, its success is primarily based on perfect knowledge and usage of all existing information about protein function and structure. To achieve that, protein engineering is supported by plenty of bioinformatic tools and analysis. The goal of this project is to create a new tool for protein engineering that would enable researchers to identificate related proteins with modified function in still growing biological databases. The tool is designed as an automated workflow of existing bioinformatic analyses that leads to identification of proteins with the same type of enzymatic function, but with slightly modified properties - primarily in terms of selectivity, reaction speed and stability.
29
Derevyanko, Georgy. "Structure-based algorithms for protein-protein interactions". Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENY070/document.
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Les phénotypes de tous les organismes vivants connus sont déterminés par les interactions compliquées entre les protéines produites dans ces organismes. La compréhension des réponses des organismes aux stimuli externes ou internes est basée sur la compréhension des interactions des protéines individuelles et des structures de ses complexes. La prédiction d'un complexe de deux ou plus protéines est le problème du domaine du docking protéine-protéine. Les algorithmes du docking ont habituellement deux étapes majeurs: recherche 6D exhaustive suivi par le scoring. Dans ce travail, nous avons contribués aux deus étapes sus indiquées. Nous avons développés le nouvel algorithme pour la recherche 6D exhaustive, HermiteFit. Cela est basé sur la décomposition des fonctions 3D en base Hermite. Nous avons implémenté cet algorithme dans le programme pour le fitting (l'ajustement des donnés) des cartes de densité électronique de résolution faible. Nous avons montrés qu'il surpasse les algorithmes existants en terme de temps par point tandis qu'il maintient la même précision du modèle sortant. Nous avons aussi développés la nouvelle approche de calculation de la fonction du scoring, qui est basé sur les arguments logique simples et qui évite la calculation ambiguë de l'état de référence. Nous avons comparés cela aux fonctions de scoring existantes avec l'aide du docking protéines-protéines benchmarks bien connues. Enfin, nous avons développés une approche permettant l'inclusion des interactions eau-protéine à la fonction du scoring et nous avons validés notre méthode pendant le CAPRI (Critical Assessment of Protein Interactions) tour 47The phenotype of every known living organism is determined mainly by the complicated interactions between the proteins produced in this organism. Understanding the orchestration of the organismal responses to the external or internal stimuli is based on the understanding of the interactions of individual proteins and their complexes structures. The prediction of a complex of two or more proteins is the problem of the protein-protein docking field. Docking algorithms usually have two major steps: exhaustive 6D rigid-body search followed by the scoring. In this work we made contribution to both of these steps. We developed a novel algorithm for 6D exhaustive search, HermiteFit. It is based on Hermite decomposition of 3D functions into the Hermite basis. We implemented this algorithm in the program for fitting low-resolution electron density maps. We showed that it outperforms existing algorithms in terms of time-per-point while maintaining the same output model accuracy. We also developed a novel approach to computation of a scoring function, which is based on simple logical arguments and avoids an ambiguous computation of the reference state. We compared it to the existing scoring functions on the widely used protein-protein docking benchmarks. Finally, we developed an approach to include water-protein interactions into the scoring functions and validated our method during the Critical Assessment of Protein Interactions round 47
30
Gao, Wei. "Characterization of protein interactors of Arabidopsis acyl-coenzyme a-binding protein 2". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43223837.
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31
Strasser, Rona. "Protein-protein interactions of receptors LdPEX5 and LPEX7 with PTS1 and PTS2 cargo proteins, and with glycosomal docking protein LdPEX14 for protein import into «Leishmania donovani»". Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=122960.
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A unique subcellular structure found in Leishmania donovani is the glycosome. This organelle compartmentalizes the enzymatic machinery required for multiple metabolic pathways, including glycolysis. Correct targeting of glycosomal enzymes is essential for parasite viability. Proteins targeted to the glycosome have either a C-terminal PTS1 or N-terminal PTS2 topogenic signal sequence, which is recognized by cytosolic receptors LdPEX5 or LPEX7, respectively. These cargo-loaded receptors interact with the peroxin protein LdPEX14, located on the cytosolic face of the glycosomal membrane, an event required for import of the cargo proteins into the glycosomal lumen. However, the complete glycosomal protein import pathway has not been fully elucidated. This work has been undertaken to better understand the protein-protein interactions involved in the trafficking of cargo proteins across the glycosomal membrane.The cytosolic fraction from L. donovani parasites was used to determine protein-protein interactions of receptors LdPEX5 and LPEX7. Size exclusion chromatography, isoelectric focusing, and affinity pull-downs showed that in the cytosol these receptors form large heterologous PTS1-LdPEX5-LPEX7-PTS2 complexes. Purified glycosomes were used to evaluate the effects of receptor-cargo complexes on glycosomal LdPEX14 conformation. Limited trypsin proteolysis showed that interaction of receptor-cargo complexes with native LdPEX14 protected this protein from digestion, whereas native LdPEX14 alone was highly sensitive to proteolysis. Protection was not dependent on membrane integrity as disruption of the lipid bilayer did not alter the effect of trypsin on these proteins. Native gel electrophoresis showed that native LdPEX14 forms large ~800 kDa complexes; however, when associated with receptor-cargo complexes the molecular weight of LdPEX14 complexes increased to ~1200 kDa. Alkaline carbonate extractions showed that native LdPEX14 acts like a peripheral glycosomal membrane protein; however loading with receptor-cargo complexes caused LdPEX14 to behave like an integral membrane protein. Furthermore, membrane insertion of LdPEX14 drove insertion of LdPEX5 and LPEX7 into the glycosomal membrane. Receptor-cargo complex association causes LdPEX14 to undergo a conformational change resulting in deeper membrane insertion and increase in complex size.Purification of recombinant LPEX7 was hampered by its association with bacterial chaperone protein GroEL. A refolding technique was developed to purify LPEX7 from inclusion bodies free of bacterial proteins. Far Western and protein-protein affinity assays showed that refolded LPEX7 specifically bound PTS2 proteins as both a monomer and a dimer, the co-receptor LdPEX5, and LdPEX14. Mapping of the interaction domains on LPEX7 showed that LPEX7-PTS2 interaction required the entire receptor protein, while LdPEX5 and LdPEX14 interaction motifs were situated in the N-terminal region of LPEX7.There are metabolites in glycosomes that are not imported via the peroxin based glycosomal import pathway but by glycosomal membrane transporters. L-arginine is one such metabolite; it is the substrate for the PTS1 glycosomal enzyme arginase, which catalyses the first step in the polyamine biosynthetic pathway. L-arginine is scavenged from the extracellular milieu and by the L-arginine transporter, amino acid permease 3 (LdAAP3). Subcellular fractionation showed that LdAAP3 localized to both the plasma and glycosomal membranes. Furthermore, L. donovani promastigotes were capable of sensing the L-arginine levels in the media and upregulated LdAAP3 expression on the plasma and glycosome membrane in the absence of L-arginine. These studies provide evidence that metabolite specific transporters are present on the glycosomal membrane.Together these studies contribute to the elucidation of glycosomal function in Leishmania donovani, and a better understanding of some of the mechanisms required for glycosomal import.Le glycosome est une structure subcellulaire unique qui se trouve dans le parasite Leishmania donovani. Cette organelle compartimente la machinerie enzymatique requise pour de multiples voies métaboliques, y compris la glycolyse. Le bon ciblage des enzymes du glycosome est essentiel pour la viabilité du parasite. Les protéines ciblées pour le glycosome ont une séquence signal topogénique, un PTS1 C-terminale ou un PTS2 N-terminale, qui est reconnue par les récepteurs cytosoliques, le LdPEX5 ou le LPEX7, respectivement. Ces complexes de récepteurs chargés s'interagissent avec la protéine LdPEX14, située du côté cytosolique de la membrane glycosomale, un événement requis pour le transport des protéines à travers la membrane du glycosome. Cependant, la voie complète d'importation de protéines glycosomales n'a pas été totalement élucidée. Ce travail a été entrepris pour mieux comprendre ces interactions protéine-protéine.La fraction cytosolique des parasites L.donovani a été utilisée pour déterminer les interactions protéine-protéine des récepteurs LdPEX5 et LPEX7. La chromatographie d'exclusion de taille, la focalisation isoélectrique, et les interactions d'affinité proteine-proteine ont montré que, dans les cytosols, ces récepteurs forment des grands complexes hétérologues. Les glycosomes purifiés ont été utilisés pour évaluer l'effet des complexes récepteur sur la conformation du LdPEX14. Une protéolyse limitée a montré que l'interaction du LdPEX14 chargé avec les complexes récepteur l'à protèger de la digestion à la surface de la membrane. L'électrophorèse sur gel natif a montré que le LdPEX14 forme des grands complexes de ~ 800 kDa et que lorsqu'il est associé à des complexes récepteur, le poids moléculaire des complexes LdPEX14 passe à ~ 1200 kDa. Les extractions avec le carbonate alcalin a déterminé que le LdPEX14 seul s'agit comme une protéine périphérique; mais son chargement avec des complexes récepteur l'entrainer à s'agir comme une protéine membranaire intégrale. L'insertion de LdPEX14 dans la membrane du glycosome conduit à l'insertion du LdPEX5 et LPEX7 dans la membrane aussi. L'association des complexes récepteur à causer LdPEX14 à subir un changement de conformation causant l'insertion profonde dans la membrane et l'augmentation de la taille des complexes.La purification du récepteur LPEX7 recombinante été entravée par son association avec la protéine chaperonne bactérienne GroEL. Une technique de repliement a été développé pour purifier LPEX7 en évitant l'association de protéines bactériennes. Les techniques de Far Western et d'affinité protéine-protéine ont montré que ce LPEX7 replier est spécifiquement associé à des protéines PTS2, le co-récepteur LdPEX5, et le LdPEX14. La cartographie des domaines d'interaction de LPEX7 a montré que l'interaction LPEX7-PTS2 nécessit le LPEX7 entière, alors que les motifs d'interaction avec LdPEX5 et LdPEX14 étaient situés dans sa région N-terminale.Il y a des métabolites glycosomal qui ne sont pas importés par la voie de l'importation glycosomale, mais par des transporteurs membranaires du glycosome. L-arginine est un de ces métabolites, substrat de l'enzyme glycosomale PTS1 arginase. L-arginine est récupéré dans le milieu extracellulaire par son transporteur, LdAAP3. Un fractionnement subcellulaire a été utilisés pour séparer les membranes plasmiques des glycosomes, et LdAAP3 a été localisé sur les deux membranes. De plus, des promastigotes de L. donovani sont capable de detecter le niveau de L-arginine dans le millieu, ce qui provoque une régulation positive de l'expression de LdAAP3 à la fois dans la membrane plasmique et dans la membrane du glycosome. Ces études fournissent des preuves que des transporteurs de métabolites spécifique sont présent dans la membrane du glycosome.Ensemble, ces études contribuent à l'élucidation de la fonction glycosomale de Leishmania donovani, et une meilleure compréhension de certains mécanismes nécessaires pour l'importation glycosomale.
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Jones, Susan. "Protein-protein interactions". Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338952.
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Lam, Wai Kwan. "Investigation of interaction between solube adenylyl cyclase and p34SEI-1 /". View abstract or full-text, 2010. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202010%20LAM.
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Smits, Callum, e n/a. "Structures of the pro-survival protein A1 in complex with BH3-domain peptides". University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20071218.131743.
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Protein:protein interactions are central to the regulation of the intrinsic programmed cell death (apoptosis) pathway. Opposing members of the Bcl-2 family of proteins, which have distinct sequence features, interact with each other on the outer mitochondrial membrane to regulate apoptosis. Pro-survival proteins such as Bcl-2, Bcl-x[L], Bcl-w, Mcl-1 and A1 protect cells from apoptosis and contain up to four regions of homology to Bcl-2 (Bcl-2 homology domains 1 - 4, BH1-4). Pro-apoptotic BH3-only proteins such as Bim, Puma, Noxa, Bad, Bmf, and Bid promote apoptosis by interacting with and inactivating pro-survival proteins, and contain just the BH3-domain. The pro-apoptotic proteins Bax and Bak are essential for apoptosis and contain three regions of homology to Bcl-2 (the BH1-, BH2- and BH3-domains).
In this study, two different sets of interactions involving pro-survival proteins were investigated. Initially, the pro-apoptotic protein Bnip3 was examined to determine if it was a mitochondrial anchor for the pro-survival protein Bcl-w. Secondly, to characterise the interactions between a pro-survival protein and different BH3-domains, structures were solved of the pro-survival protein A1 in complex with four different BH3-domains.
In the structure of Bcl-w, the hydrophobic C-terminus is bound to its own BH3-domain binding groove. This location of the C-terminus is consistent with the observation that Bcl-w is only loosely associated with the outer mitochondrial membrane in healthy cells. Upon interaction of Bcl-w with a BH3-domain, Bcl-w becomes tightly associated with the mitochondrial membrane, presumably due to displacement of the C-terminal residues by the BH3-only protein. In healthy cells it has been suggested that Bcl-w is associated with the membrane due to an interaction with an unidentified membrane protein, which preliminary experiments suggested may be Bnip3. Protein interaction experiments performed in vitro and in vivo did not reveal an interaction between Bnip3 and Bcl-w.
It was originally thought that each pro-apoptotic BH3-only protein could interact with all pro-survival proteins. However, it has recently become clear that there is selectivity within the pathway suggesting functional groupings. Bim and Puma behave as originally predicted and can interact with all pro-survival proteins and are potent killers. In contrast, Noxa and Bad interact with distinct subsets of pro-survival proteins. Noxa only binds Mcl-1 and A1, while Bad binds Bcl-2, Bcl-x[L] and Bcl-w. As a result, either Noxa or Bad acting alone is a weak killer, but together they are potent. Other BH3-only proteins bind tightly to some pro-survival proteins and weakly to others.
The diversity that exists between BH3-domain sequences precludes sequence-based identification of the determinants of specificity. In this study, crystal structures of A1:Puma BH3-domain, A1:Bmf BH3-domain, A1:Bak BH3-domain and A1:Bid BH3-domain complexes have been solved. Differences identified between these structures explain some of the variation in affinities observed in pro-survival protein:BH3-domain complexes. These observations, in combination with published data, suggest that BH3-domains bind weakly when the optimal interactions with conserved residues cannot be formed. Additionally, differences were observed in the A1:Bak BH3-domain structure that may be functionally important for the regulation of Bak.
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Björkholm, Patrik. "Protein Interactions from the Molecular to the Domain Level". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-101795.
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The basic unit of life is the cell, from single-cell bacteria to the largest creatures on the planet. All cells have DNA, which contains the blueprint for proteins. This information is transported in the form of messenger RNA from the genome to ribosomes where proteins are produced. Proteins are the main functional constituents of the cell, they usually have one or several functions and are the main actors in almost all essential biological processes. Proteins are what make the cell alive. Proteins are found as solitary units or as part of large complexes. Proteins can be found in all parts of the cell, the most common place being the cytoplasm, a central space in all cells. They are also commonly found integrated into or attached to various membranes. Membranes define the cell architecture. Proteins integrated into the membrane have a wide number of responsibilities: they are the gatekeepers of the cell, they secrete cellular waste products, and many of them are receptors and enzymes. The main focus of this thesis is the study of protein interactions, from the molecular level up to the protein domain level. In paper I use reoccurring local protein structures to try and predict what sections of a protein interacts with another part using only sequence information. In papers II and III we use a randomization approach on a membrane protein motif that we know interacts with a sphingomyelin lipid to find other candidate proteins that interact with sphingolipids. These are then experimentally verified as sphingolipid-binding. In the last paper, paper IV, we look at how protein domain interaction networks overlap and can be evaluated.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Wong, Chung Kai. "The DIX domain protein Ccd1 inhibits JNK activation by axin and dishevelled through distinct mechanisms /". View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20WONG.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 60-68). Also available in electronic version. Access restricted to campus users.
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Chivian, Dylan Casey. "Application of information from homologous proteins for the prediction of protein structure /". Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9264.
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Fang, Lin. "Mechanism of client protein binding by heat shock protein 90 /". view abstract or download file of text, 2006. http://proquest.umi.com/pqdweb?did=1251819301&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2006.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115-121). Also available for download via the World Wide Web; free to University of Oregon users.
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LOBO, DENISE DA SILVEIRA. "PROTEIN-PROTEIN INTERACTION ANALYSIS OF THE DEFENSIN PSD1 FROM PISUM SATIVUM WITH NEUROSPORA CRASSA PROTEINS". PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2006. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=9447@1.
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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRODefensinas de planta, componentes inatos do sistema imune das plantas, são peptídeos antifúngicos, catiônicos, com estrutura primária rica em cisteína. Evidência dada pela literatura demonstrou que trechos de esfingolipídios complexos na membrana dos fungos, contendo manosildiinositolfosforilceramida e glicosilceramida, são sítios de ligação seletivos para as defensinas de planta isoladas de Dahlia merckii e Raphanus sativus, respectivamente. Entretanto, desconhece-se se as defensinas de planta interagem direta ou indiretamente com alvos intracelulares dos fungos. A fim de identificar interações físicas e diretas do tipo proteína- proteína, um sistema de duplo-híbrido, em levedura, baseado no fator de transcrição GAL4, foi construído utilizando-se como isca, a defensina da planta Pisum sativum, Psd1 (Pisum sativum defensin 1). Proteínas alvos, capazes de interagirem com o peptídeo Psd1, foram detectadas através do rastreamento de uma biblioteca de cDNA do fungo Neurospora crassa. Do resultado deste rastreamento, nove dentre quinze candidatos, selecionados pelo método do duplo-híbrido, foram identificados como proteínas nucleares da N. crassa. Um clone, detectado com alta freqüência neste rastreamento, apresentou homologia de seqüência com a proteína ciclina F, relacionada com o controle do ciclo celular. O ensaio de co-purificação utilizando a proteína conjugada a glutationa S-transferase (GST) validou in vitro o resultado obtido pelo sistema duplohíbrido. Análise por microscopia de fluorescência da Psd1, conjugada a FITC, e, dos núcleos do fungo Fusarium solani, marcados com DAPI, demonstrou in vivo a co-localização da defensina de planta Psd1 com os núcleos do fungo. Para pesquisar o modo de ação da Psd1 ao nível do ciclo celular, utilizou-se o modelo multicelular da retina de ratos neonatais, em desenvolvimento. Neste modelo, a migração nuclear intercinética, correlacionada com as transições de fase de S para M do ciclo celular, foi observada na presença da Psd1. Verificouse que Psd1 impediu a migração nuclear em neuroblastos, parando o ciclo celular na transição de S para G2. Estes resultados revelaram modos de ação da defensina de planta Psd1 sobre a fisiologia nuclear.
Plant defensins, innate components of the plant immune system, are cationic, antifungal peptides, with a cysteine- rich primary structure. Evidence from the literature demonstrated that fungus membrane patches containing complex sphingolipids, mannosyldiinositolphosphorylceramide and glucosylceramides, are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. However, whether the plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify direct physical protein-protein interactions, a GAL4-based yeast two-hybrid system was constructed, using the plant peptide, Pisum sativum defensin 1 (Psd1), as the bait protein. Target proteins, capable of interacting with the bait Psd1, were detected by screening a Neurospora crassa cDNA library. In this screening, nine out of fifteen two-hybrid candidates were identified as N. crassa nuclear proteins. One clone, detected with high frequency in the screening, presented sequence similarity to a N. crassa cyclin F, related to the cell cycle control. The GST pull- down co purification assay corroborated this two-hybrid result in vitro. Fluorescence microscopy analysis of FITC- conjugated Psd1 and DAPI-stained Fusarium solani nuclei demonstrated in vivo the co-localization of the plant peptide Psd1 and the fungus nuclei. We used the developing retina of neonatal rats as a multicellular model to study Psd1 mode of action at the cell cycle level. In this model, we observed in vivo the interkinetic nuclear migration, correlated to the transitions from S to M-phase of the cell cycle, in the presence of the Psd1 peptide. It was shown that Psd1 impaired nuclear migration of neuroblasts by arresting the cell cycle at the S to G2- phase transition. These results revealed modes of action of the plant defensin Psd1 upon the nuclear physiology.
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Slavoff, Sarah Ann. "Enzyme-mediated labeling of proteins and protein-protein interactions in vitro and in living cells". Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/62060.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
The E. coli biotin ligase enzyme, BirA, has been previously used by the Ting research group for site-specific labeling of peptide-tagged cell surface proteins. We sought to expand the utility of biotin ligase-mediated labeling to functional group handles, including azides and alkynes, for bio-orthogonal chemistry. Since the BirA and its point mutants were unable to ligate these probes to an acceptor peptide, we screened biotin ligases from multiple species to identify more permissive enzymes. We determined that the Pyrococcus horikoshii biotin ligase utilizes an azide-bearing biotin analog and that the Saccharomyces cerevisiae biotin ligase can utilize an alkyne-functionalized biotin analog. We subsequently demonstrated that the azidefunctionalized biotin analog can be derivatized with a phosphine probe via the Staudinger ligation. We next turned to the goal of delivering quantum dots to the cytosol of living cells, which in the future may permit intracellular single-molecule imaging. We investigated viral methods of delivery, but found that our protocol caused quantum dots to be trapped in endocytic vesicles. We then validated previous reports that the pore-forming toxin streptolysin 0 be used to deliver quantum dots to the cytosol of living cells. Lipoic acid ligase, or LpIA, has been previously applied to site-specific protein labeling of peptide-tagged proteins using small molecule probes including lipoic acid and coumarin fluorophores. We utilized LpIA and its substrate, the LAP peptide, to create sensors for proteinprotein interactions. If LpIA is fused to one protein and LAP is fused to another, only when the two proteins interact do LpIA and LAP come into proximity, allowing probe ligation onto the peptide to occur as a readout of the interaction. We demonstrate that proximity-dependent coumarin ligation detects protein-protein interactions in living mammalian cells with extremely low background, a signal-to-background ratio of at least 5:1, and sufficiently fast kinetics to label interactions with a half-life of at least 1 minute. The reporter quantitatively responds to subpopulations of interacting proteins, allowing dissociation constants to be measured. Coumarin fluorescence accurately reports the subcellular localization of the interaction under study. Finally, we applied proximity-dependent coumarin ligation to imaging of the interaction of PSD-95 and neuroligin-1, two proteins involved in synaptic maturation, in neurons.
by Sarah Ann Slavoff.
Ph.D.
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Nauli, Sehat. "Folding kinetics and redesign of Peptostreptococcal protein L and G /". Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/9237.
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Crane, Jennine Marie. "Characterization of two modes of interaction between the chaperone SecB and its binding partners". Free to MU Campus, others may purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3144410.
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Smetana, Juliana Helena Costa. "Caracterização das proteinas TIPRL e alfa4, reguladores de fosfatases 2A". [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317177.
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Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-13T09:08:00Z (GMT). No. of bitstreams: 1 Smetana_JulianaHelenaCosta_D.pdf: 8660811 bytes, checksum: cb33e97d4c49fdce1e29094a2f6089cc (MD5) Previous issue date: 2009
Resumo: As células respondem constantemente a uma enorme variedade de estímulos, que são interpretados e integrados por meio de redes de sinalização, dando origem a uma resposta biológica. Defeitos nesses circuitos são a causa de diversas doenças, incluindo muitos, se não todos os tipos de câncer. As fosfatases, enzimas que removem grupamentos fosfato dos substratos de quinases, dependem principalmente de subunidades regulatórias para definir sua especificidade. As fosfatases do tipo 2A constituem a subfamília PPP, que é formada por PP2A, PP4 e PP6. PP2A é a principal fosfatase solúvel de fosfosserina e fosfotreonina em células animais e é encontrada predominantemente como uma holoenzima formada por uma subunidade catalítica (C), uma subunidade regulatória (B, B', B'' ou B''') e uma de ancoragem (PR65/A). Em levedura, as fosfatases 2A desempenham um importante papel na via da quinase TOR, o que ocorre por meio da proteína essencial Tap42. A proteína Tip41 foi identificada como um parceiro de interação de Tap42 e regulador da via da quinase TOR em levedura. A homóloga de Tap42 em mamíferos, chamada de a4, está envolvida na regulação de diversos processos celulares, como diferenciação, desenvolvimento, migração celular e apoptose, por meio de seu papel conservado de regulador de fosfatases 2A. A homóloga em mamíferos de Tip41, chamada TIPRL, é uma proteína ainda pouco caracterizada. Este trabalho teve como objetivo analisar a função das proteínas a4 e TIPRL humanas e esclarecer seu papel na regulação de fosfatases 2A. A caracterização estrutural de a4 e Tap42, usando dados de SAXS, dicroísmo circular e proteólise limitada, mostrou que essas proteínas apresentam um domínio N-terminal compacto formado por a-hélices e um domínio C-terminal desestruturado. Em uma triagem de interações com a proteína TIPRL humana, identificamos as fosfatases PP2Ac, PP4c e PP6c como seus parceiros de interação, assim como os fatores de transcrição MafB e TAF10. Ao contrário do esperado a partir do modelo de levedura, a4 e TIPRL não interagem diretamente, mas formam um complexo ternário com PP2Ac. Uma triagem de substratos de fosfatases 2A regulador por TIPRL identificou os fatores de splicing SF2/ASF e SF2p32. Nossos resultados sugerem um modelo estrutural para a regulação das fosfatases 2A por a4 e mostram que TIPRL é um novo regulador comum dessas fosfatases com funções na regulação da expressão gênica.
Abstract: Cells respond constantly to a variety of stimuli, which are interpreted and integrated through signaling networks, giving rise to biological responses. Defects in this circuitry are a cause of many diseases, including cancer. Protein phosphatases are enzymes which remove phosphate groups from kinase substrates, relying mainly on regulatory subunits for their substrate specificity. Type 2A phosphatases belong to the PPP subfamily, which is formed by PP2A, PP4 and PP6. PP2A is the major soluble serine/threonine phosphatase in animal cells and is found predominantly as a heterotrimer composed of a catalytic (C), a regulatory (B, B', B'' or B''') and a scaffold (PR65/A) subunit. Type 2A phosphatases play a major role in the yeast TOR signaling pathway through their interaction with the essential protein Tap42. Tip41 was identified as a Tap42 interacting protein and regulator of the TOR pathway. a4, the mammalian orthologue of Tap42, regulates many cellular processes such as differentiation, development, cell migration and apoptosis as a conserved type 2A phosphatase regulator. TIPRL, the mammalian orthologue of Tip41, is still poorly characterized. The objective of the present work was to analyse the function of a4 and TIPRL and improve the understanding of their role as type 2A phosphatase regulators. The structural characterization of a4 using SAXS analyses, circular dichroism and limited proteolysis, showed that these proteins are formed by an a-helical N-terminal domain and an unfolded C-terminal domain. A screen for TIPRL interacting proteins identified PP2Ac, PP4c and PP6c and also the transcription factors MafB and TAF10. Unlike their yeast conterparts, a4 and TIPRL do not interact directly, but rather form a ternary complex with PP2A. A search for type 2A phosphatase substrates regulated by TIPRL identified the splicing factor SF2/ASF and its regulatory protein SF2p32. Our results suggest a structural model for the regulation of type 2A phosphatases by a4 and show that TIPRL is a novel common regulator of these phosphatases which functions in regulation of gene expression.
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Wang, Huachun. "Protein phosphorylation regulation in Arabidopsis". Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/5896.
Texto completo da fonteResumo:
Thesis (Ph. D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on July 18, 2008) Vita. Includes bibliographical references.
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Tse, Man-kit. "Structural characterization and domain dissection of human XAF1 protein, and application of solvent-exposed-amide spectroscopy in mapping protein-protein interface". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43223801.
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Kwan, Ann H. Y. "Protein design based on a PHD scaffold". Connect to full text, 2004. http://setis.library.usyd.edu.au/adt/public_html/adt-NU/public/adt-NU20041202.102526/index.html.
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Thesis (Ph. D.)--School of Molecular and Microbial Biosciences, Faculty of Science, University of Sydney, 2004.Chapter headings on separately inserted unnumbered cream coloured leaves. Bibliography: leaves 122-135.
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Dehzangi, Abdollah. "Protein Fold Recognition Using Segmentation-based Features". Thesis, Griffith University, 2015. http://hdl.handle.net/10072/366423.
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Proteins are considered to be one of the most important biological macromolecules and play a wide range of vital roles in most biological interactions. Therefore, determining how they function is an important task in biology and biomedical science. Protein fold recognition is defined as assigning a given protein to a fold (among a finite number of folds) that represents its functionality as well as its major tertiary structure. Despite all the efforts made over the last two decades, finding an effective computational approach to solve this problem still remains challenging for computational biology and bioinformatics.
In this study we enhance the protein fold prediction accuracy by employing evolutionary and structural information for feature extraction. Based on our previously proposed feature extraction techniques to extract physicochemical-based features, we develop segmented distribution and segmented auto-covariance feature extraction methods to extract local evolutionary and structural information. By applying an SVM to our extracted features, we enhance the protein fold prediction accuracy up to 7.2% better than the best result found in the literature. In conclusion, we develop several segmentation-based feature extraction techniques that enable us to extract local discriminatory information for protein fold recognition better than previously proposed approaches to achieve this goal.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Integrated and Intelligent Systems
Science, Environment, Engineering and Technology
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Maschkowitz, Gregor [Verfasser]. "Untersuchung der Protein-Protein-Interaktion der Proteine ppUL35 und ppUL35A des humanen Cytomegalievirus mit dem zellulären Protein Sorting Nexin 5 / Gregor Maschkowitz". Kiel : Universitätsbibliothek Kiel, 2017. http://d-nb.info/1138979988/34.
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Cooper, Simon T. "PAX6 protein-protein interactions". Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/29070.
Texto completo da fonteResumo:
The gene PAX6 is located on chromosome 11 (11p13) and encodes a transcription factor (PAX6) that is expressed early in development. The PAX6 protein is expressed in the developing eye, regions of the brain, central nervous system (CNS), nasal epithelium and pancreas. PAX6 is best known for its role eye development with heterozygous mutations causing congenital ocular malformations. However, it must be remembered that PAX6 has multiple functions in the brain including specification of neuronal subtypes and axon guidance. There is growing understanding of the role of PAX6 as a transcription factor during development, and many of its DNA targets have recently been defined. However, almost nothing is known about the proteins with which PAX6 interacts. In the initial stage of my research I identified a conserved region consisting of the final 32 amino acids of the PST (proline, serine and threonine rich) domain of PAX6. Based on sequence homology and secondary structure predictions I classed this region as a novel domain, the ‘C terminal domain’. Next I used the yeast 2-hybrid system to investigate possible PAX6 protein interactions. By screening a mouse brain cDNA library with the C terminal domain and whole PST domain, I identified three novel and interesting interactors, HOMER3, DNCL1 and TRIM11. I re-confirmed these interactions in a pairwise manner using the yeast 2-hybrid system, and I showed that the C terminal domain was vital for the interactions between PAX6 and HOMER3 or DNCL1. Furthermore, certain C terminal mutations that are known to cause ocular malformations in patients are also sufficient to reduce or abolish these interactions. I attempted to further characterise the interactions by co-immunoprecipitation. However, this was not possible due to technical difficulties.
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Shevchenko, Anna. "Characterization of Protein Complexes and Protein Interaction Networks by Mass Spectrometry". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2004. http://nbn-resolving.de/urn:nbn:de:swb:14-1101913240562-37411.
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The major goal of this study was to develop an experimental proteomics approach for deciphering protein complexes and protein interaction networks in the budding and fission yeasts. Key steps of the employed analytical routine, including the purification of complexes and mass spectrometric identification of their subunits, were investigated in detail. Archiving, storage and handling of polyacylamide gels, visualization of protein bands and their effect on the efficiency of in-gel digestion and mass spectrometric identification of proteins were quantitatively evaluated. It was further demonstrated that a combination of several mass spectrometric techniques based on MALDI and ES ionization provided complementary data and enabled comprehensive characterization of protein digests. The optimized analytical procedures were employed in deciphering protein complexes and protein interaction networks in the budding and fission yeasts. A combination of Tandem Affinity Purification (TAP) and mass spectrometric identification of gel separated protein subunits is generic and robust strategy that provided accurate and reproducible data. The evaluation of TAP success rate, reproducibility and typical protein background presented in this work is based on TAP tagging and immunoprecepitation of 75 genes in S. cerevisiae and 22 in S. pombe. The molecular composition of characterized protein complexes was compared with protein-protein interactions uncovered by other established methods, such as yeast two hybrid screens or proteome-wide purification of protein complexes. We found that repetitive purification of protein complexes using different subunits as baits is crucially important for confident charting of proteomic environments. Accurate dissection of individual protein complexes and identification of their proteomic hyperlinks enabled to consider proteomic environments in the phylogenetic perspective and paved the way to reliable projection of proteomics data obtained in lower eukaryotic model organisms to higher eukaryotes, including humans.