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1
Kulman, John David. "Transmembrane Gla proteins /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9271.
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Doak, David G. "Peptide models of transmembrane proteins." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359445.
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Käll, Lukas. "Predicting transmembrane topology and signal peptides with hidden Markov models /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-719-7/.
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Garrow, Andrew Gordon. "Search algorithms for transmembrane beta-barrel proteins." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427773.
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Peters, Christoph. "Topology Prediction of α-Helical Transmembrane Proteins." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-129061.
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Membrane proteins fulfil a number of tasks in cells, including signalling, cell-cell interaction, and the transportation of molecules. The prominence of these tasks makes membrane proteins an important target for clinical drugs. Because of the decreasing price of sequencing, the number of sequences known is increasing at such a rate that manual annotations cannot compete. Here, topology prediction is a way to provide additional information. It predicts the location and number of transmembrane helices in the protein and the orientation inside the membrane. An important factor to detect transmembrane helices is their hydrophobicity, which can be calculated using dedicated scales. In the first paper, we studied the difference between several hydrophobicity scales and evaluated their performance. We showed that while they appear to be similar, their performance for topology prediction differs significantly. The better performing scales appear to measure the probability of amino acids to be within a transmembrane helix, instead of just being located in a hydrophobic environment. Around 20% of the transmembrane helices are too hydrophilic to explain their insertion with hydrophobicity alone. These are referred to as marginally hydrophobic helices. In the second paper, we studied three of these helices experimentally and performed an analysis on membrane proteins. The experiments show that for all three helices positive charges on the N-terminal side of the subsequent helix are important to insert, but only two need the subsequent helix. Additionally, the analysis shows that not only the N-terminal helices are more hydrophobic, but also the C-terminal transmembrane helices. In Paper III, the finding from the second paper was used to improve the topology prediction. By extending our hidden Markov model with N- and C-terminal helix states, we were able to set stricter cut-offs. This improved the general topology prediction and in particular miss-prediction in large N- and C-terminal domains, as well the separation between transmembrane and non-transmembrane proteins. Lastly, we contribute several new features to our consensus topology predictor, TOPCONS. We added states for the detection of signal peptides to its hidden Markov model and thus reduce the over-prediction of transmembrane helices. With a new method for the generation of profile files, it is possible to increase the size of the database used to find homologous proteins and decrease the running time by 75%.
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Batrakou, Dzmitry G. "Nuclear envelope transmembrane proteins in differentiation systems." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9981.
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Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA replication and repair and development. Several recent proteomic studies uncovered a great variety of nuclear envelope transmembrane proteins (NETs). Diseases associated with several nuclear envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin, bones. Many NETs of the inner nuclear membrane have been shown to interact with chromatin, suggesting that their influencing gene expression might explain NET roles in disease. This work is focused on finding novel interactions of NETs with chromatin. First, SUN2 post-translational modifications were analysed and the effect of phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton was tested by overexpression. However, no obvious changes were found. Second, several tissue-preferential NETs were tested in an adipocyte differentiation system. NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in chromosome positioning that occur during adipocyte differentiation when NET29 is normally induced. Post-translational modifications of NET29 are likely to play a vital role in this process because a phospho-null mutant dominantly blocked chromosome repositioning. The effect of over-expression and down-regulation of NET29 on transcription was tested and results suggest that NET29 negatively regulates expression of myogenic genes during adipogenesis. This thesis is split into six chapters. Chapter I is an overview of the nuclear envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed description of methods used in this study. Chapter III focuses on post-translational modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter IV and V deal with a novel nuclear envelope transmembrane protein and its role in adipogenesis. Finally, the last chapter includes a discussion and recommended future directions.
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Kelm, Sebastian. "Structural modelling of transmembrane domains." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b4c9fba9-ee25-469b-8baf-b7c1d70c9d05.
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Membrane proteins represent about one third of all known vertebrate proteins and over half of the current drug targets. Knowledge of their three-dimensional (3D) structure is worth millions of pounds to the pharmaceutical industry. Yet experimental structure elucidation of membrane proteins is a slow and expensive process. In the absence of experimental data, computational modelling tools can be used to close the gap between the numbers of known protein sequences and structures. However, currently available structure prediction tools were developed with globular soluble proteins in mind and perform poorly on membrane proteins. This thesis describes the development of a modelling approach able to predict accurately the structure of transmembrane domains of proteins. In this thesis we build a template-based modelling framework especially for membrane proteins, which uses membrane protein-specific information to inform the modelling process.Firstly, we develop a tool to accurately determine a given membrane protein structure's orientation within the membrane. We offer an analysis of the preferred substitution patterns within the membrane, as opposed to non-membrane environments, and how these differences influence the structures observed. This information is then used to build a set of tools that produce better sequence alignments of membrane proteins, compared to previously available methods, as well as more accurate predictions of their 3D structures. Each chapter describes one new piece of software or information and uses the tools and knowledge described in previous chapters to build up to a complete accurate model of a transmembrane domain.
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Crick, Duncan James. "Solution NMR studies of seven-transmembrane helix proteins." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708906.
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Berthoumieu, Olivia. "Single molecule studies of seven transmembrane domain proteins." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:ff7ae71d-5481-4523-812b-2128fe32f5fc.
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This work aimed at studying biophysical properties of two membrane proteins, one of potential nanotechnological use, bacteriorhodopsin, and one potential drug target, the NTS1 neurotensin receptor, at the single molecule scale. Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) of the halophilic organism Halobacterium salinarium. It is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of its retinal chromophore. Its stability, as well as its photoactivity remaining in dry protein layers, has made BR an attractive material for biomolecular devices. Numerous studies have been published on this topic; however, they have all used BR within the PM, on relatively large (µm-wide) surfaces. Here, conducting-probe atomic force microscopy (C-AFM) analysis was performed after removing most of the membrane lipids. For the first time, it was shown that the molecular conductance of BR can be reversibly photoswitched with predictable wavelength sensitivity. Intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine which, combined with partial delipidation, generated protein trimers homogenously orientated on the surface. Numerous controls using biophysical (SPR, ellipsometry, Kelvin-probe AFM) and chemical (photocurrent, cyclic voltammetry) techniques confirmed the wavelength specificity of the photoswitch, the anchoring role of the mutation and the homogenous orientation of the protein on the gold surface. Neurotensin is a brain and gastrointestinal 13 amino acid peptide acting as a neuromodulator in the central nervous system and as a hormone in the periphery. Its wide range of biological activities is primarily mediated through its binding to the neurotensin type 1 receptor (NTS1). NTS1 expressed in E.coli was purified and inserted into 100 nm brain polar lipid liposomes in a conformation which retained its ligand-binding capabilities. Initial AFM characterisation was performed as a prelude for ligand-receptor interaction studies, including high resolution imaging, force spectroscopy and solid state NMR approaches.
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Abd, Halim Khairul Bariyyah. "Molecular dynamics simulation studies of transmembrane signalling proteins." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:bc9e1e0e-433c-4adb-8374-1065eac0f37e.
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Receptor tyrosine kinases (RTKs) are a major class of cell surface receptors, important in cell signalling events associated with a variety of functions. High-throughput (HTP), coarse-grained molecular dynamics (CG-MD) simulations have been used to investigate the dimerization of the transmembrane (TM) domain of selected RTKs, including epidermal growth factor receptor (EGFR) and muscle-specific kinase (MuSK). EGFR activation requires not only a specific TM dimer interface, but also a proper orientation of its juxtamembrane (JM) domain. Phosphatidylinositol 4,5-bisphosphate (PIP2) is known to abolish EGFR phosphorylation through interaction with basic residues within the JM domain. Here, a multiscale approach was used to investigate anionic lipid clustering around the TM-JM junction and how such clustering is modulated by the mutation of basic residues. The simulations demonstrated that PIP2 may help stabilize the JM-A antiparallel dimer, which may in turn help stabilize TM domain helix packing of the N-terminal dimerization motif. A proximal TM domain residue has been implicated in the inhibition of ganglioside GM3 in phase-separated membranes. Here, CG simulations were used to explore the dynamic behaviour of the EGFR TM domain dimer in GM3-containing and GM3-depleted bilayers designed to resemble lipid-disordered (Ld) and phase-separated (Ld/Lo) membranes. The simulations suggest that the presence of GM3 in Ld/Lo bilayers can disrupt and destabilize the TM dimer, which helps to explain why GM3 may favour monomeric EGFR in vivo. To gain insights into the dynamic nature of the intact EGFR, a nearly complete EGFR dimer was modelled using available structural data and embedded in an asymmetric compositional complex bilayer, which resembles the mammalian plasma membrane. The results demonstrated the dynamic nature of the EGFR ectodomain and its predicted interactions with lipids in the local bilayer. Strong protein-lipid interactions, as well as lipid-lipid interactions, affect the local clustering of lipids and the diffusion of lipids in the vicinity of the protein on both leaflets.
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Suzuki, Takayuki. "Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F." Kyoto University, 2014. http://hdl.handle.net/2433/188693.
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Qureshi, Tabussom. "Studying Transmembrane Helix Interactions in SDS micelles." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34417.
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The importance of interactions between transmembrane domains of integral membrane proteins has been well-established in a range of essential cellular functions. Most integral membrane proteins also possess regions that lie on the exterior of the membrane that may influence the ability of these transmembrane domains to interact. We sought to test this hypothesis by quantifying the energetics of transmembrane helix self-association in the absence and presence of an amphipathic helix that can bind to the membrane surface. The model chosen for this study was the major coat protein (MCP) of M13 bacteriophage, which has an N-terminal amphipathic helix linked to its single transmembrane segment via a flexible linker. Dimerization of both full-length MCP and a peptide containing only the transmembrane domain (MCPTM) was studied by solution NMR in SDS micelles. We found that there was an increase in the apparent dimerization affinity in the absence of the N-terminal helix. However, this increase in apparent affinity could be attributed to differences in detergent-binding properties of the two polypeptides in monomeric versus dimeric states when the empty micelle was considered to be a participant in the dimer dissociation.
Preliminary results from the integral membrane protein, p7 of the hepatitis C virus are also presented in this thesis. It has been demonstrated that p7 enhances viral infectivity and accumulation, and that this function may require oligomerization in the membrane. While we encountered limitations due to challenges in the generation of sufficient quantities of pure p7 samples, we were able to perform circular dichroism spectroscopy under conditions that may favor different oligomeric states. These studies suggest that there is a change in the degree of helicity upon oligomerization, and suggest that SDS could be a suitable system to characterize the interactions of the p7 oligomer in the future.
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Adcock, Stewart Alan. "Computer simulation of membrane bound molecules." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249194.
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Jacobson, Leslie William. "Antibodies to the human muscle acetylcholine receptor : their specificity and function in the foetus." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299108.
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Hagelbäck, Johan, and Kenny Svensson. "Locating transmembrane domains in protein sequences." Thesis, Blekinge Tekniska Högskola, Institutionen för programvaruteknik och datavetenskap, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:bth-2752.
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We have developed a new approach for locating transmembrane domains in protein sequences based on hydrophobicity analysis and backpropagation neural network or k-nearest-neighbor as classifiers. Our system was able to locate over 98% of the transmembrane domains and the total accuracy including overpredictions was above 95%.
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Jenn, Robert. "A systematic analysis of human transmembrane E3-RING proteins." Thesis, University of Liverpool, 2011. http://livrepository.liverpool.ac.uk/5913/.
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The reversible covalent conjugation of the small highly conserved ubiquitin protein modifier to selective substrates plays central roles in countless proteolytic and non-proteolytic cellular functions. Substrate protein ubiquitination is co-ordinated by the sequential activity of three distinct classes of proteins: (i) E1-activating enzymes, (ii) E2-conjugating enzymes, and (iii) E3-protein ligases. Really Interesting New Gene (RING) proteins represent the largest family of E3-proteins comprising over half of predicted human E3-ligases. As such, E3-RING proteins play pivotal roles in controlling both specificity and functionality within the ubiquitin system. E3-RING proteins function as catalytically inactive molecular scaffolds that position Ub~E2 and substrate proteins in close proximity for ubiquitination to occur. Within the active ligase complex, E3-RING proteins and E2 conjugating enzymes are believed to select protein substrate(s) and the form of conjugated ubiquitin upon them, respectively. Whilst E3-RING/E2 partners have been investigated in recent HTP screen approaches, a key area of data paucity exists for integral membrane E3-RING (TM-E3-RING) proteins. As such, high throughput yeast-two-hybrid assays were performed for the entire complement of TM-E3-RING proteins and E2-conjugating enzymes. A broad subset of TM-E3-RING/E2 positive and negative Y2H interactions was re-tested in secondary luciferase protein complementation assays (PCAs), which increased confidence in Y2H-derived interactions and extended network coverage. Data from these studies was collated with previously published binary TM-E3-RING/E2 interaction data to provide a high-confidence TM-E3-RING/E2 network consisting of 312 unique binary interactions. In vitro auto-ubiquitination assays were employed to assign functional activity to TM-E3-RING/E2 protein pairs, revealing high verification rates for both positive and negative Y2H or PCA binary interaction data. Furthermore, novel trends in the generation of different forms of ubiquitin modifications were identified between selective TM-E3-RING/E2 pairs. Finally, Y2H screens were also performed to identify TM-E3-RING dimerization events, which represent an emerging theme in ubiquitin system regulation. In total 71 TM-E3-RING/TM-E3-RING interactions were reported demonstrating high incidence of these binding events. Novel data was combined with known interactions to generate a TM-E3-RING network containing >500 binary interactions, encompassing both components of the core ubiquitin cascade and non-ubiquitome proteins. This TM-E3-RINGcentric network provides a valuable tool for the investigation of specificity and regulation of TM-E3-RING proteins and specific ubiquitin cascades.
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Schroeder, Michael, Annalisa Marsico, Andreas Henschel, Christof Winter, Anne Tuukkanen, Boris Vassilev, and Kerstin Scheubert. "Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-177368.
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Background
A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology.
Results
We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94%) appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1) a dimer interface motif found in voltage-gated chloride channels, (2) a proton transfer motif found in heme-copper oxidases, and (3) a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b.
Conclusions
Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.
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Cassel, Marika. "Studies on the Conformation of Transmembrane Polypeptides in Membrane Proteins." Doctoral thesis, Stockholm : Deptartment of Biochemistry & Biophysics, Stockholm University, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-759.
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Hurwitz, N. "Developing a novel method for homology detection of transmembrane proteins." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1386009/.
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Analysis of the complete genomic sequences for several organisms indicates that 20-25% of all genes code for transmembrane proteins (Jones, 1998, Wallin and von Heijne, 1998), yet only a very small number of transmembrane 3D structures are known. Hence, it is of great importance to develop theoretical methods capable of predicting transmembrane protein structure and function based on protein sequence alone. To address this, we sought to devise a systematic and high throughput method for identifying homologous transmembrane proteins. Since protein structure is more evolutionarily conserved than amino acid sequence, we predicted that adding structural information to simple sequence alignment would improve homology detection of transmembrane proteins. In the present work, we describe development of a search method that combines sequence alignment with structural information. In our method the initial sequence alignment searches are performed using PSI-BLAST. Then profiles derived from the multiple sequence alignments are input into a neural network, developed in this work to predict which transmembrane residues are buried (core of the helix-bundle) or exposed (to the lipid environment). A maximum accuracy of 86% was achieved. Moreover, for almost half of the query set, the predicted residue orientation was more than 70% accurate. In the last step of the work presented here, the predicted helix locations, residue orientations and loop length scores are added to the PSI-BLAST E-value, to create a ‘combined’ classifier. A linear equation was built for calculating the 'combined’ classifier score. Our method was evaluated using two databases of proteins: Pfam and GPCRDB. The Pfam database was chosen, as transmembrane proteins in this database have been classified into various families. GPCRDB was employed as this database, though narrow, is well-studied and maintained. Before building the ‘combined’ classifier, PSI-BLAST sequence alignment was benchmarked using the Pfam database. We found that our 'combined’ classifier, as compared to a classifier based solely on PSI-BLAST, resulted in more true positives with less false positives when tested using GPCRDB and could differentiate between GPCRDB families. However, our ‘combined’ classifier did not improve homology detection when searching transmembrane proteins from the Pfam database. A comparison of our ‘combined’ classifier method with two other published methods suggested that profile-profile based searches could be more powerful than profile-sequence based searches, even after the addition of structural information as described here. In light of our study, we propose that combining structural information with profile-profile sequence alignment into a 'combined’ classifier could result in a search method superior to any existing ones for detecting homologous transmembrane proteins.
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Holdbrook, Daniel. "Molecular dynamics studies of transmembrane proteins within complex lipid environments." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/366965/.
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The interactions between lipids and proteins are crucial for many cellular processes. Typically, the nature of these interactions is studied in simple model lipid bilayers, which lack the complexity and heterogeneity of in vivo systems. Thus, this thesis investigates the impact of the lipid bilayer composition on protein dynamics and function. Both coarse grain and atomistic molecular dynamics simulations have been used to model membranes that contain lipid compositions approximating those found in vivo. The influence of these complex lipid environments on the dynamics of α-helical and β-barrel membrane protein is investigated. In particular, coarse grained simulations of a bilayer composed of a complex mixture of lipids, representing the Golgi apparatus, were used to identify preferential interactions of a helical transmembrane peptide with PIP2 lipids. Furthermore, atomistic molecular dynamics simulations have been used to identify several behaviour altering interactions between lipopolysaccharide, which is a key component of the Gram-negative bacterial outer membrane, and two outer membrane proteins, Hia and FecA. Lastly, coarse grained unilamellar vesicles, containing a complex mixture of phospholipids, were simulated in order to investigate protein aggregation and the short-term anomalous diffusion of lipids.
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Le, Thanh Phu. "Nuclear envelope transmembrane proteins as mediators of tissue-specific diseases." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28905.
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Many tissue-restricted diseases are linked to mutations in lamins and nuclear envelope transmembrane proteins (NETs). How these mutations in ubiquitously expressed proteins cause such defined diseases is still unknown. It is hypothesized that tissue restricted NETs that are partners of the nuclear lamins/existing linked proteins mediate tissue-specific disease pathologies. Proteomic studies have identified many tissue restricted NETs with effects on the cytoskeleton, gene positioning and regulation. This study investigates potential roles of candidate NETs in mediating tissue restricted disease pathology and their interactions with known factors such as emerin and lamins, mutations in which have been linked to a variety of tissue-specific dystrophies. This study looks into candidate tissue-specific NETs distribution in human tissues and in vitro using a solid phase binding assay to study candidate NETs interactions. I confirmed the tissue-specificity of the candidate NETs in human and mouse tissue sections but did not find clear reproducible distribution of these NETs in patient tissue biopsy. One postulate is that NETs bind WT lamin for localisation and/or function and disruption of this interaction leads to disease. Using a solid phase binding assay approach to study NETs/lamin interactions, we demonstrate that Tmem120a, an adipocyte-specific NET binds WT lamin but has a reduced Bmax when tested for binding against a lipodstrophy causing lamin mutant (R482Q and G465D). This is consistent with the hypothesis that tissue-specific NET partners might mediate tissue-specific disease pathology in lamin-linked nuclearenvelopathies.
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Mauk, Andrew W. "Determination of the structure of the magainin II transmembrane channel." Diss., Georgia Institute of Technology, 1999. http://hdl.handle.net/1853/11708.
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Schroeder, Michael, Annalisa Marsico, Andreas Henschel, Christof Winter, Anne Tuukkanen, Boris Vassilev, and Kerstin Scheubert. "Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins." BioMed Central, 2010. https://tud.qucosa.de/id/qucosa%3A28887.
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Background
A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology.
Results
We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94%) appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1) a dimer interface motif found in voltage-gated chloride channels, (2) a proton transfer motif found in heme-copper oxidases, and (3) a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b.
Conclusions
Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.
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Nilsson, IngMarie. "Conformational properties of transmembrane polypeptide segments in the ER membrane /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3613-7/.
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Hedin, Linnea E. "Intra- and intermolecular interactions in proteins : Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-42856.
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Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.At the time of the doctoral defense the following publications were not published and had a status as follows: Paper 2: In press; Paper 4 Manuscript.
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D'Rozario, Robert S. G. "Conformational dynamics of proline-containing transmembrane helices." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670181.
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Thevenin, Damien. "Roles of transmembrane domains in the folding and assembly of the adenosine A2A receptor." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 170 p, 2007. http://proquest.umi.com/pqdweb?did=1260822171&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Renner, Lars. "Polymer Supported Lipid Bilayer Membranes for the Integration of Transmembrane Proteins." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1241457489091-02157.
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This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biologyDie vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen
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Lu, Jennifer. "Interferon-induced transmembrane proteins inhibit human immunodeficiency virus type 1 replication." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95120.
Full textAbstract:
Viral infection triggers production of interferon (IFN) that in turn leads to the expression of genes known as IFN-stimulated genes (ISGs), some of which possess antiviral activities. Previous studies have shown that IFN suppresses the replication of human immunodeficiency virus type I (HIV-1). While several ISGs have been linked to this specific antiviral activity with well-defined inhibitory mechanisms, others remain to be investigated. With the purpose of identifying novel ISGs capable of inhibiting HIV-1 replication, we have performed a shRNA screen of the genes upregulated by IFN in SupT1 cells. This study reports three ISGs, known as interferon-induced transmembrane proteins 1, 2 and 3 (IFITM1, 2 and 3), that substantially inhibit HIV-1 replication in SupT1 cells. Further studies suggest that HIV-1 entry is impaired. Collectively, these findings identify a small family of cellular restriction factors that serve as a barrier to HIV-1 entry into the host cell.Suite à une infection virale, les interférons (IFNs) sont produites et servent à induire l'expression de certains gènes, appelés gènes stimulés par l'interféron (ISGs), dont certains possèdent des effets antivirales. Plusieurs études ont démontré que l'IFN possède la capacité d'inhiber la réplication virale du virus de l'immunodéficience humaine de type I (VIH-1). Tandis que certains ISGs ont été associés à une activité antivirale spécifique avec un mécanisme d'action bien défini, d'autres ISGs sont moins bien caractérisés. Dans le but d'identifier de nouveaux ISGs responsables d'inhiber la réplication virale du VIH-1, nous avons réalisé un criblage par shRNA des gènes régulés par l'IFN dans les cellules SupT1. Cette étude rapporte trois ISGs, appelés «interferon-induced transmembrane proteins 1, 2 et 3» (IFITM1, 2, et 3), dont l'expression dans les cellules SupT1 peut inhiber la réplication virale du VIH-1 de façon significative. Les résultats indiquent que ces protéines agissent au niveau de l'entrée du virus dans la cellule. Collectivement, cette étude a identifié une famille de facteur de restriction cellulaire qui agit comme barrière pour prévenir l'entrée du VIH-1 dans la cellule hôte.
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King, Gavin W. "Investigating helix-helix interactions in the transmembrane domains of membrane proteins." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/3157/.
Full textAbstract:
Helix-helix interactions between membrane-spanning transmembrane (TM) domains have been shown to drive the assembly of α-helical membrane proteins within biological membranes. However, the rules that determine these interactions are not yet fully understood, despite such interactions being found in an increasing number of proteins. Recent work has implicated TM domain interactions in the formation of the protein complex Ii-MHC, formed from the association of Major Histocompatibility Complex Class II (MHC) and the MHC-associated-Invariant Chain (Ii) proteins. Following biosynthesis, three MHC α/βheterodimers bind to the Ii homotrimer to form a nonameric Ii-MHC complex within the endoplasmic reticulum. This is a critical step in the export of MHC molecules to the antigen presentation system and hence the activation of an immune response to a pathogen. In this study we have explored the TM domain interactions within the Ii-MHC complex. Results from in vivo and in vitro experiments revealed the TM domains of the α- and β-chains of MHC have a propensity to self-associate into homo-dimers and to associate with one another to form hetero-dimers. Highly conserved GxxxG motifs (known to drive dimerization) were implicated in these interactions. The TM domain of Ii was confirmed to self-associate to form trimers by in vivo and in vitro methods, but surprisingly also displayed additional oligomeric states suggesting the interaction is not as specific as was previously thought. Furthermore, we show that in vivo, the TM domain of Ii can associate with those of the α- and β-chains of MHC, whilst in vitro methods suggested Ii preferentially binds to α-chains. Collectively, these findings strongly suggest that the TM domains of Ii and MHC have a role to play in the assembly of the Ii-MHC complex, and hence the very important process of antigen presentation. Additionally, in this study we have undertaken development of NMR spectroscopy methods that have the potential to increase our understanding of not only the Ii-MHC complex, but protein-protein interactions in general.
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Tran, Thuong Van Du. "Modeling and predicting super-secondary structures of transmembrane beta-barrel proteins." Phd thesis, Ecole Polytechnique X, 2011. http://tel.archives-ouvertes.fr/tel-00647947.
Full textAbstract:
Les protéines transmembranaires canaux-β (TMBs) se trouvent dans les membranes externes des bactéries à Gram négatif, des mitochondries ainsi que des chloroplastes. Elles traversent entièrement la membrane cellulaire et exercent différentes fonctions importantes. Vu qu'il y a un petit nombre des structures des TMBs déterminées, en raison des difficultés avec les méthodes expérimentales, il est douteux que ces approches puis- sent bien trouver et prédire les TMBs qui ne sont pas homologues avec celles connues. Nous construisons un modèle de graphe pour la classification et la prédiction de structures super-secondaires permutées des TMBs à partir de leur séquence d'acides aminés, en se basant sur la minimisation d'énergie. Le modèle ne dépend essentiellement pas de l'apprentissage. Les algorithmes sont rapides, robustes avec des performances com- parables à celles des meilleures méthodes actuelles qui utilisent l'apprentissage. Cette méthode peut être donc utile pour le screening des génomes. Outre la performance de prédiction et de classification, cette étude donne une vue plus profonde de la structure des TMBs en tenant compte des contraintes physicochimiques des membranes biologiques. Les structures permutées prédites peuvent aussi aider à mieux comprendre le mécanisme du repliement des TMBs.
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Phatak, Mukta. "Lipid Accessibility Prediction and Identification of Functional Hotspots in Transmembrane Proteins." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1267631564.
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Muller, Pari-Sima Sieglinde. "Characterisation of fibronectin leucine-rich repeat transmembrane proteins during mouse embryonic development." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540289.
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Runwal, Gautam. "The study of two transmembrane autophagy proteins and the autophagy receptor, p62." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290149.
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Autophagy is an evolutionarily conserved process across eukaryotes that is responsible for degradation of cargo such as aggregate-prone proteins, pathogens, damaged organelles, macromolecules etc. via its delivery to lysosomes. The process is known to involve the formation of a double-membraned structure, called autophagosome, that engulfs the cargo destined for degradation and delivers its contents by fusing with lysosomes. This process involves several proteins at its core which include two transmembrane proteins, ATG9 and VMP1. While ATG9 and VMP1 has been discovered for about a decade and half, the trafficking and function of these proteins remain relatively unclear. My work in this thesis identifies and characterises a novel trafficking route for ATG9 and VMP1 and shows that both these proteins traffic via the dynamin-independent ARF6-associated pathway. Moreover, I also show that these proteins physically interact with each other. In addition, the tools developed during these studies helped me identify a new role for the most common autophagy receptor protein, p62. I show that p62 can specifically associate with and sequester LC3-I in autophagy-impaired cells (ATG9 and ATG16 null cells) leading to formation of LC3-positive structures that can be misinterpreted as mature autophagosomes. Perturbations in the levels of p62 were seen to affect the formation of these LC3-positive structures in cells. This observation, therefore, questions the reliability of LC3-immunofluorescence assays in autophagy-impaired cells as method of assessing autophagy and points towards the homeostatic function played by p62 in autophagy-impaired cells.
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Möller, Steffen. "An environment for consistent sequence annotation and its application to transmembrane proteins." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619671.
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Nordström, Karl J. V. "Characterization and Evolution of Transmembrane Proteins with Focus on G-protein coupled receptors in Pre-vertebrate Species." Doctoral thesis, Uppsala universitet, Funktionell farmakologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121696.
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G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. GPCRs are instrumental for hormonal and neurotransmitter signalling and are important in all major physiological systems of the body. Paper I describes the repertoire of GPCRs in Branchiostoma floridae, which is one of the species most closely related species to vertebrates. Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Paper II contains studies of the Adhesion, Methuselah and Secretin GPCR families in nine genomes. The Adhesion GPCRs are the most complex gene family among GPCRs with large genomic size, multiple introns and a fascinating flora of functional domains. Phylogenetic analysis showed Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family among the groups in the Adhesion family, which was also supported by splice site setup and conserved motifs. Paper III examines the repertoire of human transmembrane proteins. These form key nodes in mediating the cell’s interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins. We identified 6,718 human membrane proteins and classified the majority of them into 234 families of which 151 belong to the three major functional groups; Receptors (63 groups, 1,352 members), Transporters (89 groups, 817 members) or Enzymes (7 groups, 533 members). In addition, 74 Miscellaneous groups were shown to include 697 members. Paper IV clarifies the hierarchy of the main families and evolutionary origin of majority of the metazoan GPCR families. Overall, it suggests common decent of at least 97% of the GPCRs sequences found in humans, including all the main families.
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Qiu, Fei-Hua. "Proto-oncogene c-kit : structure and relationship to the transmembrane receptor kinases /." Access full-text from WCMC, 1989. http://proquest.umi.com/pqdweb?did=744572251&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Chetwynd, Alan. "Computational studies of transmembrane helix insertion and association." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:440da098-5bd6-4fcb-8396-645517ac2122.
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Membrane proteins perform a variety of functions essential for the viability of the cell, including transport and signalling across the membrane. Most membrane proteins are formed from bundles of transmembrane helices. In this thesis molecular dynamics simulations have been used to investigate helix insertion into bilayers and helix association within bilayers. The potentials of mean force for the insertion of helices derived from the cystic fibrosis transmembrane conductance regulator into lipid bilayers were calculated using coarse-grained molecular dynamics simulations. The results showed that the insertion free energy increased with helix length and bilayer hydrophobic width. The insertion free energies obtained were significantly larger than comparable quantities obtained from translocon- mediated insertion experiments, consistent with a variety of previous studies. The implications of this observation for the interpretation of in vivo translocon-mediated insertion experiments, and the function of the translocon, are discussed. Coarse-grained and atomistic molecular dynamics simulations of the transmembrane region of the receptor tyrosine kinase EphA1 suggested that the transmembrane helix dimer was most stable when interacting via the glycine zipper motif, in agreement with a structure obtained by NMR spectroscopy. Coarse-grained simulations of the transmembrane region of EphA2 suggested that the dimer has two stable orientations, interacting via a glycine zipper or a heptad motif. Both structures showed right-handed dimers, although an NMR structure of the transmembrane region of EphA2 shows a left-handed dimer interacting via the heptad motif. Both structures obtained from coarse-grained simulations proved unstable when simulated at an atomistic level of detail. The potentials of mean force for dissociating the EphA1 and EphA2 dimers were calcu- lated using coarse-grained molecular dynamics calculations. Convergence of the detailed structure of the profiles was not conclusively shown, although association free energies cal- culated from the profiles were consistent over a variety of simulation times. The association free energies were slightly larger than experimental values obtained for comparable sys- tems, but consistent with similar computational calculations previously reported. However, direct comparisons are difficult owing to the influence of environmental factors on reported association free energies. The potential of mean force profiles showed that the interaction via the glycine zipper motif for EphA1 was significantly more stable than any other confor- mation. For EphA2 the potential of mean force profiles suggested that interaction via the glycine zipper and heptad motifs both provided stable or metastable conformations, with the interaction via the glycine zipper motif probably at least as stable as that via the heptad motif.
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Kho, Yik Shing. "Identification and characterization of a novel family of transmembrane and coiled-coil proteins /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BICH%202008%20KHO.
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Mokrab, Younes. "Insights into sequence-structure relationship in helical transmembrane proteins : application to comparative modeling." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611915.
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Zhang, Zhihui. "Assembly and Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator and Associated Proteins." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/101.
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Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which is believed to affect the folding and stability of CFTR. The misfolded ΔF508-CFTR undergoes ER associated degradation (ERAD), causing the failure of ΔF508-CFTR trafficking to the cell surface. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. In this dissertation, real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however the amount of channel in each vesicle remained the same. Therefore, a 2 step therapeutic approach might be an ideal treatment for CF. The first step would be composed of a compound that mimics the mechanism of stabilization provided by low temperature or the I539T mutation, while the second step would be VX-809 or a similar corrector compound. These studies suggest that understanding how low temperature and second site suppressors alter ΔF508-CFTR could be key to the development of future therapeutics for the effective treatment of CF.
The precise pathophysiology of cystic fibrosis is not well studied. The involvement of another transport protein, epithelial sodium channel (ENaC), makes the situation more complicated. ENaC and CFTR are colocalized on the apical surface of epithelia cells. With our fluorescence microscopy techniques, we explored the effects of CFTR on the residence time of ENaC on the cell membrane. A reliable approach measuring the half-life of protein on the cell membrane is required for this study. We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Photoconversion of Dendra2 works as a pulse chase experiment by monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. The half-life of the protein on the plasma membrane was calculated upon imaging over time and quantifying the change in red fluorescence. Our method provides a unique opportunity to observe real-time protein turnover at the single cell level without addition of protein synthesis inhibitors. This technique will be valuable for the future protein half-life study.
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Lang, Tiange. "Evolution of transmembrane and gel-forming mucins studied with bioinformatic methods /." Göteborg : The Sahlgrenska Academy at Göteborg University, Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, 2007. http://hdl.handle.net/2077/7502.
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Whitehead, Thomas James. "Characterisation of chicken interferon-inducible transmembrane proteins : locus architecture, gene expression and viral restriction." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10058159/.
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Interferon-inducible transmembrane (IFITM) proteins are host cell derived restriction factors. Mammalian IFITM proteins have been shown to confer antiviral resistance when challenged with a diverse range of both enveloped and non-enveloped viruses. Little characterisation has been undertaken to date with the specific aim of elucidating their function and the antiviral properties of the chicken IFITM (chIFITM) gene family. The chIFITM gene family contains four genes located within a 17kb region on Gallus gallus chromosome 5. Currently there is little information available about the sequence diversity of these genes, their expression profiles or the role that they may play in restricting avian viral pathogens. Data presented in this thesis outlines a novel DNA pull-down sequencing technique which has allowed for the generation of a high quality contiguous reference sequence, alongside targeted sequencing of chicken cell lines and ex vivo cell cultures. Studies in this thesis have established that the chIFITMs are interferon stimulated and have characterized their upregulation in response to viral challenge with influenza A virus (IAV) in ovo, in vivo and in vitro, alongside other avian viruses. Stably-overexpressing DF-1 (immortalized chick embryo fibroblast) cells that express chIFITM1, 2, 3 and 3MUT (C71/71A) have been generated. These cell lines have been challenged with avian viruses including diverse strains of IAV and infectious bronchitis virus (IBV) and this data demonstrates that the chIFITMs are able to restrict avian viruses in vitro. Moreover, novel interactions have been identified which may help to uncover a possible mechanism of action. Global food security and protection of livestock from infectious agents remains a key priority, both in the United Kingdom and Internationally. This study examines the role of chIFITM proteins during viral infections and highlights one potential method of safeguarding the poultry industry, ensuring continuity of global food security.
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Hallstrom, Kelly N. "The Epithelial Transmembrane Protein PERP Is Required for Inflammatory Responses to S. typhimurium Infection: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/807.
Full textAbstract:
Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea.
Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity.
In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation.
Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.
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Hallstrom, Kelly N. "The Epithelial Transmembrane Protein PERP Is Required for Inflammatory Responses to S. typhimurium Infection: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/807.
Full textAbstract:
Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea.
Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity.
In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation.
Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.
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Sittaramane, Vinoth Chandrasekhar Anand. "Role of transmembrane protein strabismus in motor neuron migration in the zebrafish hindbrain." Diss., Columbia, Mo. : University of Missouri--Columbia, 2008. http://hdl.handle.net/10355/6623.
Full textAbstract:
Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Anand Chandrasekhar. Vita. Includes bibliographical references.
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Damaghi, Mehdi. "Characterizing the Functional and Folding Mechanism of β-barrel Transmembrane Proteins Using Atomic Force Microscope." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-114947.
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Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010).
Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent.
In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process.
We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG.
Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.
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Goldstein, Rebecca F. "Protein interaction and cell surface trafficking differences between wild-type and [Delta]F508 cystic fibrosis transmembrane conductance regulator." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007p/goldstein.pdf.
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Simpson, Janet Elizabeth. "The cystic fibrosis transmembrane conductance regulator and acid-base transporters of the murine duodenum." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4391.
Full textAbstract:
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. Vita. "August 2006" Includes bibliographical references.
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Calado, Botelho Salomé. "Translocation of proteins into and across the bacterial and mitochondrial inner membranes." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-83234.
Full textAbstract:
Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening. This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively. These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane. A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease. Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 1: Manuscript; Paper nr. 4: Manuscript