Dissertations / Theses: 'Ligands (Biochemistry) Membrane proteins'

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Leng, Ying. "Neuron-ligand pathfinding on surfaces modified by laminin and laminin-derived peptides." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 78 p, 2006. http://proquest.umi.com/pqdweb?did=1203562381&sid=8&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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Millman, Jonathan Scott Andrews David. "Characterization of membrane-binding by FtsY, the prokaryote SRP receptor /." *McMaster only, 2002.

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Ma, Jerome H. Y. "Atomistic studies of the dynamics of P-glycoprotein and its ligands." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:e2e2bbe0-d4ae-4351-b339-c8e02ef3d3d9.

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A signifficant obstacle facing the healthcare industry is the phenomenon of multidrug resistance (MDR) in which a cell acquires simultaneous resistance to many unrelated drugs that it has never been exposed to. At the molecular level, MDR can be characterised by a reduction of intracellular drug levels due to their active efflux by multidrug transporters such as P-glycoprotein (Pgp). Pgp is able to efflux a phenomenally wide variety of chemically unrelated drugs and causal relationships have been established between its expression and the acquisition of MDR to numerous anticancer and central nervous system (CNS) drugs. There has thus been much effort to understand the molecular biology of Pgp and how it functions. However, many aspects of its functioning remain unclear. From a drug discovery viewpoint, we have yet to fully understand what features make some drugs susceptible to Pgp-mediated efflux (substrates) and what makes others able to inhibit Pgp function (inhibitors). From a mechanistic viewpoint, it is still uncertain what the exact nature of Pgp's binding site is, the role of ATP binding and hydrolysis in transport and how both of these interplay with ligand binding. The work presented in this thesis attempts to answer these questions from two perspectives. Firstly the mouse Pgp crystal structure [PDB 3G60] was used as a unique starting point for molecular dynamics (MD) simulations to characterise the dynamics and conformational exibility of Pgp, properties believed to be integral to its function. The simulations revealed Pgp to be a highly dynamic molecule at both its transmembrane (TM) and nucleotide binding domains (NBDs). The latter exhibited a conformational asymmetry that supports the Constant Contact model of ATPase activity. In the presence of the Pgp substrate, daunorubicin, the NBDs exhibited tighter asymmetric dimerisation leading to increased affinity for ATP. In contrast, the presence of the Pgp inhibitor, QZ59-RRR led to NBD conformational changes that reduced their affinity for ATP. Thus providing an appealing mechanism for how QZ59-RRR inhibits Pgp ATPase activity. MD simulation was also used to provide atomic-detail interpretations of multiple binding stoichiometries of drug and lipid molecules observed by collaborator-led mass spectrometry experiments. This also provided opportunity to validate the Pgp simulations against novel experimental data. The second strand of the thesis explored the membrane permeation dynamics of CNS therapeutics in order to identify differences in protonation states, conformations, orientations and membrane localisation that might distinguish those that are Pgp substrates and from those that are not. These properties were studied using complementary MD simulation and nuclear magnetic resonance (NMR) techniques. The simulations revealed a novel set of criteria that in uence the likelihoodof a drug to 'flip-flop' across a membrane, a behaviour that may make drugs more susceptible to Pgp efflux. These observations were broadly consistent with the NMR experiments. However, the NMR data also highlighted limitations in the simulation approaches used in this thesis and emphasised the need to also consider the kinetics of permeation in addition to its thermodynamics.

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Christie, Shaun Michael. "Elucidation of Membrane Protein Interactions Under Native and Ligand Stimulated Conditions Using Fluorescence Correlation Spectroscopy." University of Akron / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=akron1594383686413803.

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Sahai, Michelle Asha. "Computational studies of ligand-water mediated interactions in ionotropic glutamate receptors." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b86d2f5a-3554-44c0-b985-5693241369ec.

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Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.

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Rapp, Mikaela. "The Ins and Outs of Membrane Proteins : Topology Studies of Bacterial Membrane Proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1330.

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Davies, R. J. "Monolayer studies on intrinsic erythrocyte membrane proteins." Thesis, University of Manchester, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356110.

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Boekel, Carolina. "Integration and topology of membrane proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-8575.

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Yue, Kevin Kin Man. "Assembly of outer membrane proteins in Escherichia coli." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257436.

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Fairbairn, L. J. "Investigations on erythrocyte membrane proteins using molecular cloning techniques." Thesis, University of Bristol, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379600.

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Chapman, Rowan Emma. "Sorting and retention of golgi-localised integral membrane proteins." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360699.

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Zhang, Dongmei. "Rotational motion and organization studies of cell membrane proteins." Thesis, Colorado State University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10137939.

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Cell membranes are dynamic structures with complex organization. The complexity of the cell membrane arises from intrinsic membrane structure, membrane microdomains within the plasma membrane and the membrane cytoskeleton. Plasma membrane receptors are integral membrane proteins with diverse structures and functions which bind specific ligands to trigger cellular responses. Due to compartmentalization of the plasma membrane and the formation of membrane microdomains, receptors are distributed non-homogeneously in the cell membrane bilayer. Both lateral and rotational diffusion of membrane receptors reflects different kinds of intermolecular interactions within the plasma membrane environment. Understanding protein diffusion within the membrane is very important to further understanding biomolecular interactions in vivo during complex biological processes including receptor-mediated signaling.

Rotational diffusion depends linearly on the in-membrane volume of the rotating proteins. Relative to lateral diffusion, rotational diffusion is a more sensitive probe of an individual molecule’s size and local environment. We have used asymmetric quantum dots (QD) to conduct imaging measurements of individual 2H3 cell Type I Fcϵ receptor rotation on timescales down to 10 msec per frame. We have also used time-tagged single photon counting measurements of individual QD to examine µsec timescales, although rapid timescales are limited by QD emission rates. In both approaches, decays of time-autocorrelation functions (TACF) for fluorescence polarization fluctuations extend into the millisecond timescale, as implied by time-resolved phosphorescence anisotropy results. Depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Imaging results demonstrate a range of rotational behavior among individual molecules. Such slow motions, not observable previously, may occur with large signaling complexes, which are important targets of study in cell biology. These slow motions appear to be a property of the membrane itself, not of the receptor state. Our results may indicate that individual mesoscale membrane regions rotate or librate with respect to the overall cell surface.

The luteinizing hormone receptor (LHR) is a seven transmembrane domain receptor and a member of the GPCR family. It is located on luteal cells, granulosa and theca cells in females. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of mammalian reproduction. LHR becomes aggregated upon binding hCG when receptors are expressed at physiological numbers. Binding of hormone to LHR leads to activation of adenylate cyclase (AC) and an increase in intracellular cyclic AMP (cAMP). ICUE3 is an Epac-based cAMP sensor with two fluorophores, cyan fluorescent protein (CFP) and the YFP variant, cpVenus, and a membrane-targeting motif which can be palmitoylated. Upon binding cAMP, ICUE3 undergoes a conformational change that separates CFP and YFP, significantly reducing FRET and thus increasing the ratio of CFP to YFP fluorescence upon excitation with an arc lamp or 405nm laser source. Hence we have investigated hLHR signal transduction using the cyclic AMP reporter probe, ICUE3. A dual wavelength emission ratio (CFP/YFP) imaging method was used to detect a conformational change in ICUE3 upon binding cAMP. This technique is useful in understanding the sequence of intercellular events following hormone binding to receptor and in particular, the time course involved in signal transduction in a single cell. Our data suggested that CHO cells expressing ICUE3 and directly treated with different concentrations of cAMP with saponin can provide a dose-dependent relationship for changes in intracellular cAMP levels. Forskolin (50µM) causes maximal activation of the intracellular cAMP and an increase in the CFP/YFP emission ratio. In CHO cells expressing both ICUE3 and hLHR-mCherry, the CFP/YFP ratio increased in cells treated with forskolin and in hCG- treated cells. In flow cytometry studies, similar results were obtained when CHO cells expressed < 60k LHR-mCherry per cell. Our results indicate that ICUE3 can provide real time information on intracellular cAMP levels, and the ICUE3 is a reliable cAMP reporter can be used to examine various aspects of LH receptor-mediated signaling.

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Pongprayoon, Prapasiri. "Molecular modelling of β-barrel outer membrane proteins." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:0ed0c22f-027e-4be1-a64c-0819888bbebc.

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In Gram-negative bacteria, the Outer membrane (OM) acts as a first barrier to screen unwanted compounds whilst enabling ions and very small solutes to diffuse into the cell. Most of nutrients and essential ions are effectively transported across a membrane via the outer membrane proteins (OMPs). The water-filled β- barrel OMPs are called porins. These pores are classified into two groups, non- specific and substrate-specific porins. Each of them has different mechanisms to facilitate its substrate translocation. To reveal the process of substrate permeation and selectivity in microscopic detail, molecular dynamics (MD) simulations and applications were performed in this thesis. The studies in this thesis focus on a series of classical porins. These proteins share similar feature where extracellular loop(s) (generally loop 3 (L3)) is folded into the middle of the pore and act as a constriction site which is important for substrate specificity and selectivity. The studies firstly concentrate on the structural properties and dynamics of the general trimeric porins, OmpC and OmpF whose sequences share 60% identity. OmpC and OmpF are found to have similar mechanism of latching loop (L2) to maintain trimeric stability. The smaller pore size allows OmpC to be more cation-selective than OmpF. Additionally, the major driving force for cation permeation in both porins is not from electrostatic properties. This differs from the phosphate-selective porin, trimeric OprP, where a phosphate diffusion depends on electrostatic interactions with positively charged pore-lining residues. The charge brush-like behavior of interior Arg and Lys residues plays a major role in phosphate selectivity. Also, the free energy profiles (PMF) reveal two key regions that are important for differentiating phosphate from other anions. The brush-like mechanism of OprP were also implanted to the simplified model pores in order to determine the possibility of transferring phosphate-selective properties of OprP to a model which may be useful for future design of nanopores. It is found that the duplication of functional residues and pore cavity can turn a model into the highly phosphate-selective pore. Importantly, the phosphate-binding affinity is dependent on the ability of the pore to interfere and occupy the hydration shell of a translocating phosphate where such ability can be maximized by an increase in sidechain flexibility. In case of uptake of more complex substrates, OpdK also employs a constriction site to select its substrate, aromatic vanillate (VNL) with total charge of -1. Unlike ion-specific porins, the free VNL is attracted by polar and aromatic interactions and sequentially directed through the periplasmic vestibule by charged residues insides the pore. The correct orientation of VNL on arrival is crucial for OpdK to recognize and enable the permeation process.

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Chauhan, Vinita. "Low density lipoprotein receptor, interaction with ligands and molecular chaperone proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ66136.pdf.

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15

Ye, Cui. "STABILITY STUDIES OF MEMBRANE PROTEINS." UKnowledge, 2014. http://uknowledge.uky.edu/chemistry_etds/33.

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The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms. The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding. The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.

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Wagner, Samuel. "From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7513.

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In both pro- and eukaryotes 20-30% of all genes encode alpha-helical transmembrane domain proteins, which act in various and often essential capacities. Notably, membrane proteins play key roles in disease and they constitute more than half of all known drug targets. The natural abundance of membrane proteins is in general too low to conveniently isolate sufficient material for functional and structural studies. Therefore, most membrane proteins have to be obtained through overexpression. Escherichia coli is one of the most successful hosts for overexpression of recombinant proteins. While the production of soluble proteins is comparably straightforward, overexpression of membrane proteins remains a challenging task. The yield of membrane localized recombinant membrane protein is usually low and inclusion body formation is a serious problem. Furthermore, membrane protein overexpression is often toxic to the host cell. Although several reasons can be postulated, the basis of these difficulties is not completely understood, preventing the design of rational strategies to improve membrane protein overexpression yields. The objective of my Ph.D. studies has been to improve membrane protein overexpression in E. coli by a) understanding membrane protein overexpression from the perspective of membrane protein biogenesis, b) systematically investigating the physiological response to overexpression of membrane proteins and c) engineering strains that are optimized for membrane protein overexpression based on insights resulting from these studies. By working toward these objectives, I was able to identify and alleviate one of the major bottlenecks of membrane protein overexpression in E. coli: saturation of the Sec-translocon could be overcome by harmonizing translation and membrane insertion of the recombinant membrane protein. This minimized the toxic effects of overexpression and thus resulted in increased membrane protein-producing biomass.

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Gee, N. S. "Studies on pig kidney microvillar membrane proteins using monoclonal antibodies." Thesis, University of Leeds, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355702.

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Wallin, Erik. "Theoretical studies of Membrane Proteins : Properties, Prediction Methods and Genome-wide analysis." Doctoral thesis, Stockholm : Univ, 1999. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-30.

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Wang, Ruiqi Rachel. "Biochemical and Structural Studies of Membrane Proteins." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10154.

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Membrane proteins live at the interface between a cell and its environment; hence, they play a variety of important physiological roles such as transmembrane transport, signal transduction, and cell adhesion. The importance of membrane proteins in biology and medicine requires that we understand their structure and function on the atomic level. In this thesis, I studied members of two different membrane protein families, namely the neuronal and keratinocyte TRPV ion channels that sense temperature changes and MP20, a member of the PMP22/EMP/MP20/claudin superfamily. Using a variety of biochemical, X-ray crystallographic and electrophysiological techniques, I addressed mechanistic questions pertaining to the regulation of thermosensitive TRPV channels by ATP and calmodulin in neurons and keratinocytes. For MP20, a protein specific for the lens of the mammalian eye, I used a vesicle assay in combination with electron microscopy (EM) to study its function, ruling out the possibility that MP20 is involved in the formation of membrane junctions. Furthermore, I made progress in expressing and crystallizing MP20 for X-ray diffraction studies. In a separate effort, I also worked on improving and expanding the use of monolayer purification and Affinity Grids, recently introduced techniques to prepare specimens for single-particle EM based on the recruitment of His-tagged proteins to nickel lipidcontaining lipid monolayers. I extended the use of these techniques by synthesizing a glutathione lipid that can be used to recruit GST-tagged proteins. A major hurdle in the use of monolayer purification techniques, however, is the extent of non-specific protein binding to the lipid monolayer. I found that incorporating PEG lipids in the monolayer appears to reduce the problem of non-specific protein binding. While it remains to be seen whether these techniques can be developed to a point at which it will be possible to recruit exclusively tagged proteins out of cell lysates, my goal is to continue to improve and expand the use of the monolayer purification and Affinity Grid techniques in hope to make single-particle EM more easily amenable to biochemists and cell biologists.

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Orwick, Marcella Christine. "Biophysical and magnetic resonance studies of membrane proteins." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:e7974f5f-a5ab-4867-aa5f-feff99716c0f.

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Bacteriorhodopsin (bR) is a 7TM membrane protein expressed in Halobacterium salinarum. Due to its stability and high expression levels, bR serves as a model for other 7TM membrane proteins. Neurotensin receptor 1 (NTS1) is a member of pharmacologically relevant G protein-coupled receptor superfamily, and is the high affinity receptor for neurotensin, a 13mer peptide that can be found in the brain, gut, and central nervous system. NTS1 is a target for Parkinson’s, Schizophrenia, and drug addiction. This thesis aims to develop pulsed magnetic resonance techniques and sample preparation forms for high resolution structural studies on 7TM proteins. In this thesis, pulsed dipolar distance electron paramagnetic resonance (EPR) methods for the study of proteins in their native membrane are established. bR is spin-labeled, and a wellresolved distance distribution is measured in excellent agreement with other structural data. Preliminary distance data for a photoexcited state of bR suggests quaternary rearrangements in the native membrane that are agreement with published AFM results. A fitting method is developed to enable measurements of systems with rapid signal decay, a common feature in reconstituted systems studied by pulsed EPR methods. A physical chemical characterization of nanosized-bilayer discs termed Lipodisqs®, and the successful incorporation of bR is presented. Lipodisqs® are formed from DMPC and a polymer that is able to solubilize DMPC vesicles into discrete particles. Lipodisqs® possess a broad phase transition with increased lipid ordering compared to a DMPC dispersion. The SMA polymer interacts with the lipid tails, but does not perturb the headgroup. BR is incorporated in the monomeric form, and EPR dynamic and distance measurements confirm that Lipodisqs® preserve the native structure of bR, whilst detergent solubilisation increases the overall mobility compared to bR in its native membrane, suggesting that Lipodisqs® serve as an excellent medium for EPR studies on 7TM membrane proteins. A cysteine-depleted mutant of active, ligand binding NTS1 is constructed. Cysteines are reintroduced at positions that may be able to monitor agonist and inverse-agonist induced conformational and dynamic changes. A spin-labeling protocol is developed, and preliminary EPR measurements are discussed. Dynamic nuclear polarization (DNP) results are presented with uniformly-¹³C-labelled bR in the PM, resulting in a DNP enhancement of 16 using the biradical nitroxide polarizing agent, TOTAPOL. DNP-enhanced solid state NMR (ssNMR) is typically carried out at cryogenic temperatures, resulting in poor spectral resolution compared to ambient temperatures. Two different forms of samples are prepared that could potentially lead to better-resolved DNP spectra. BR is reverse labelled by adding natural abundance amino acids to isotopically labelled growth medium, resulting in the partial depletion of resonance signals that may obscure and crowd the NMR spectra. A crystalline sample of bR is prepared using the LCP method for crystallization, which is to date the most successful method for the crystallization of GPCRs. In summary, the first pulsed dipolar measurements of a protein in its native membrane are shown, Lipodisqs® are characterized and found to be a suitable medium for structural and functional studies of 7 TM membrane proteins, the first preliminary EPR studies on a ligand binding GPCR are presented, and novel sample preparation techniques are developed for the nitroxide-based DNP enhancement of ssNMR data. This thesis opens up several avenues for future research into 7TM membrane proteins.

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Guzzo, Rosa M. "Sarcolemmal membrane associated proteins: Structure-function analyses and localization studies." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/29048.

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Previous work from our lab identified a novel cDNA encoding a family of tail-anchored coiled-coil integral membrane proteins termed SLMAPs (sarcolemmal membrane associated proteins) (Wigle et al., 1997). Subsequent studies determined that SLMAPs are encoded by a single gene, and alternative splicing yields three SLMAP isoforms, including two muscle-specific variants (SLMAP1, SLMAP2) and a ubiquitously expressed isoform (SLMAP3). Here, I report a series of studies designed to examine putative novel and isoform specific functions of SLMAPs in striated muscle and fibroblast cells. The tissue distribution and subcellular localizations of SLMAPs were examined in developing and adult mouse tissues in order to correlate SLMAP expression with specific physiological or developmental process(es). Immunohistochemical staining using polyclonal anti-SLMAP antibodies revealed that SLMAPs are highly expressed in developing somites and cardiac tissue. Confocal microscopy determined that SLMAPs localized within the discrete membrane structures (sarcoplasmic reticulum and T-tubules) of developing and mature skeletal and cardiac muscle, respectively. These localization studies suggest a correlative role for SLMAPs in excitation-contraction (EC) coupling mechanisms. In vivo expression of SLMAPs in pre-fusion myoblasts indicated a possible involvement in skeletal myogenesis. An additional SLMAP protein was expressed under conditions that promote differentiation in cultured myoblasts. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Protein-protein interaction assays demonstrated that the leucine zipper motifs in SLMAPs mediate SLMAP homodimer formation. Proteomic analysis further revealed that a muscle-specific SLMAP variant binds a component of the contractile apparatus (cardiac myosin heavy chain). The expression of a cardiac-specific SLMAP isoform that resides in distinct membranes, self assembles and interacts with the contractile apparatus further suggest a unique role for this molecule in excitation-contraction coupling mechanisms. Alternative splicing mechanisms generate SLMAP variants with divergent carboxyl-terminal hydrophobic segments, which target SLMAPs to different membrane compartments. Immunocytochemistry studies revealed that the expression of the first transmembrane domain directs a 6Myc-SLMAP fusion protein to the endoplasmic reticulum in COS7 cells. (Abstract shortened by UMI.)

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Sladek, Barbara. "Structural studies of integral membrane GPCR accessory proteins." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:09bf7ada-8e58-49f4-a979-bcd0cec95e8b.

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GPCR accessory proteins regulate the strength, efficiency and specificity of signal transfer upon receptor activation. Due to the inherent difficulties of studying membrane proteins in vitro and in vivo, little is known about the structure and topology of these small accessory proteins. Two examples of GPCR accessory proteins are the Melanocortin-2 receptor accessory protein (MRAP) and the Receptor-activity-modifying protein (RAMP) family. MRAP and RAMP1 are the main focus of this thesis in which they are thoroughly characterised by solution-state NMR and further biophysical techniques. The single-pass transmembrane domain protein MRAP regulates the class A GPCR melanocortin receptors. It is specifically required for trafficking the melanocortin-2-receptor from the endoplasmic reticulum to the cell surface and subsequent receptor activation. A remarkable characteristic of MRAP is its proposed native dual-topology, which leads to an antiparallel homodimeric conformation. Investigation of the biochemical and biophysical properties of MRAP revealed an α-helical transmembrane domain, and an α-helical N-terminal LD(Y/I)L-motif. Further efforts concentrated on establishing the homodimeric conformation of MRAP in vitro. RAMP1 facilitates receptor trafficking and alters the ligand specificity of the GPCR Class B receptors calcitonin receptors and calcitonin receptor-like receptors. Moreover, RAMP1 is required to act as a Calcitonin-gene-related peptide (CGRP) receptor (RAMP1). RAMP1 has been shown to form stable parallel homodimers in the absence of its cognate receptor. Its dimerisation and the possible dimerisation motif PxxxxP-motif were studied extensively. With the goal of understanding the mechanism of dimerisation and the role of GPCR accessory proteins I have used solution-state NMR in detergent micelles as my main technique. NMR provides unique possibilities for understanding the structure and dynamics of such small membrane proteins.

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Pan, Yuan. "Targeting membrane proteins to inner segments of vertebrate photoreceptors." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1720.

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Photoreceptors are highly compartmentalized neurons in the retina, and they function by detecting light and initiating signaling through the visual network. The photoreceptor contains several compartments including the outer segment (OS) which is a sensory cilium for detecting photons and the inner segment (IS) that carries out important modulatory functions via its resident channels and transporters. Those proteins are membrane proteins that function together to shape electrical properties of the cell membrane during both rest and active states. Therefore it is essential to maintain proper function of the membrane proteins in the IS. One important way to regulate the function of a membrane protein is via controlling its trafficking to ensure a proper amount of the protein in the proper cellular compartment. To date, little is known about how IS membrane protein trafficking is controlled in photoreceptors. In this study, our goal is to understand those mechanisms using cell biology and biochemistry approaches. To achieve the goal, we investigated trafficking of two unrelated IS resident proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that mediates a feedback current in photoreceptors, and the sodium potassium ATPase (NKA) which maintains the basic electrochemical property of the cell. In order to study trafficking of HCN1, we first investigated the dependence of HCN1 trafficking in photoreceptors on TRIP8b, an accessory subunit that influences trafficking of HCN1 in hippocampal neurons. By studying TRIP8b knockout mice we found that TRIP8b is dispensable for HCN1 trafficking in photoreceptors but required for maintaining the maximal expression level of HCN1. Since we revealed that HCN1 trafficking can be regulated in a cell-type specific manner, we subsequently focused on the amino acid sequence of HCN1 to identify novel trafficking signals that function in photoreceptors. By examining localization of a series of HCN1 mutants in transgenic Xenopus photoreceptors, we discovered a di-arginine ER retention motif and a leucine-based ER export motif. These two sequence motifs must function together to maintain equilibrium of HCN1 level between the endomembrane system and the cell surface. The study of HCN1 uncovered a mechanism for the photoreceptor to control membrane protein trafficking via the early secretory pathways. To reveal additional trafficking machineries in photoreceptors, we investigated trafficking of NKA. We first tested for an interaction with ankyrin, an adaptor protein that regulates NKA trafficking in epithelial cells, and found these proteins do not co-localize in photoreceptors. We then aimed to identify novel trafficking signals by studying the trafficking behavior of two NKA isozymes: NKA-α 3 and NKA-α 4. When expressed in transgenic Xenopus photoreceptors, these two proteins localize to the IS and the OS respectively. By studying localization of multiple chimeras and truncation mutants, we found that the distinct localization pattern is due to a VxP OS/ciliary targeting motif present in NKA-α 4. Since NKA-α 4 is naturally expressed in the ciliary compartment of the sperm, our finding in the photoreceptor suggests a mechanism for NKA-alpha 4 trafficking in its native environment. Overall, our studies of HCN1 and NKA together provide new insights into controlling membrane protein trafficking in photoreceptors and help establish the basics for future therapeutic intervention targeting trafficking pathways that are linked to about one third of proteins reported in retinal diseases.

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Kim, Irene. "Mechanisms of Membrane Disruption by Viral Entry Proteins." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10192.

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To enter and infect cells, viruses must overcome the barrier presented by the cell membrane. Enveloped viruses, which possess their own lipid bilayer, fuse their viral membrane with the cell membrane. Non-enveloped viruses, whose outer surface is composed of proteins, penetrate through the hydrophobic interior of the cell membrane. Viruses accomplish the processes by coupling conformational changes in viral "entry proteins" to membrane disruption. This dissertation investigates the membrane disruption mechanisms of rotavirus, a non-enveloped virus, and vesicular stomatitis virus (VSV), an enveloped virus. Rotavirus uses proteins of its outer capsid to penetrate the membrane and deliver a transcriptionally-active core particle into the cell cytoplasm. \(VP5^*\), an outer capsid protein, undergoes a foldback rearrangement that translocates three clustered hydrophobic loops by \(\sim 180^{\circ}\). This rearrangement resembles the foldback rearrangements of enveloped virus fusion proteins. In the first half of my dissertation, I show that the hydrophobicity of the \(VP5^*\) apex is required for membrane disruption during rotavirus cell entry by mutating hydrophobic residues within the loop to hydrophilic residues. One particular mutation diminishes liposome interaction by the protein, blocks membrane penetration by virus particles in cells, and reduces particle infectivity by 10,000-fold. VSV uses its fusion protein, G, to fuse at low pH. Unlike other viral fusion proteins, pH-induced conformational changes in G are reversible. In the second half of my dissertation, I measure the fusion kinetics of individual VSV particles using a single-particle fusion assay previously developed for influenza virus. I find that hemifusion by VSV consists of at least two steps, an initial step that is pH-dependent and reversible, and a second step that is pH-independent. At low pHs, the second step becomes the sole rate-limiting step. I also show that at pH 6.6, the VSV particle enters a stable intermediate state that binds tightly to membranes but does not precede to fusion. This dissertation uses a variety of experimental approaches to arrive at a more detailed understanding of how viruses use their entry proteins to either penetrate or fuse with the cell membrane.

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Farhang-Fallah, Janet Rozakis-Adcock Maria. "Cloning and characterization of PHIP, a novel protein ligand of the PH domain of IRS-1 /." *McMaster only, 2002.

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Stamp, Anna Louise Elizabeth. "Structural studies of protein - ligand interactions : potential biomedical implications." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670175.

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Tate, Christopher G. "cDNA cloning of human erythrocyte membrane proteins and studies of abnormal blood group phenotypes." Thesis, University of Bristol, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327929.

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Aslimovska, Lubica. "High resolution structural studies of membrane proteins using solid state NMR." Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:6b72463e-92cf-4ecd-bdc3-26b2f900e813.

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NMR crystallography is a new and developing area. Unlike solution state NMR, solid state NMR has the potential for structural studies of large, motionally restricted biological macromolecules, such as proteins in crystals which may, or may not, diffract. However, finding the best and the most useful sample form and geometry is still a major obstacle to rapid progress. Little has been reported about protein sample preparation for any class of protein for NMR crystallography, mainly since the availability of NMR labelled proteins is still not routine, especially for eukaryotic membrane proteins. The amino acid L-glutamate is the major excitatory neurotransmitter in the brain. Details of glutamate binding to any of its main brain or sensory receptors are not well resolved at the atomic level. In an effort to resolve the glutamate binding mechanism by solid state NMR methods, full-length taste and brain mGluR4 were expressed in E. coli, but proved to be toxic for the cells. The ligand-binding domains (LBD) of mGluR4, with various fusions for the periplasmic expression and with various fusions for expression in the cytoplasm therefore, were used. Solubilisation and then purification of the LBD from inclusion bodies is still under way, no crystals of mGluR4 for NMR were, therefore, grown. Initial NMR spectra of labelled 13C, 15N and 17O glutamate have been recorded to verify sensitivity requirements. Using homology modelling, a model for the truncated version of the ligand binding domain of mGluR4 has been constructed as a basis for designing solid state NMR experiments to probe the ligand binding site in the receptor. Bacteriorhodopsin is a large membrane protein and a model for G-protein coupled receptors (GPCRs). Spectra of bacteriorhodopsin produced in H. salinarium in purple membrane are reported here and compared to spectra of the protein crystallised from bicelles. Optimal conditions for producing spectra suitable for spectral assignment are reported as an initial step towards spectral resolution. Three differently labelled samples of bacteriorhodopsin were prepared to test the applicability of the various assignment strategies and the effects of deuteration on quality of solid state NMR spectra of a large, crystalline membrane protein.

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Guna, Alina-Ioana. "Membrane protein biosynthesis at the endoplasmic reticulum." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276678.

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The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.

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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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Keyser, Rowena J. "Identifying ligands of the C-terminal domain of cardiac expressed connexin 40 and assessing its involvement in cardiac conduction disease." Thesis, Link to online version, 2007. http://hdl.handle.net/10019/651.

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Hansson, Emil. "The ins and outs of notch ligands and downstream events /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-806-1/.

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Wyatt, Katrina. "Erythrocyte anion transporter : its interaction with cytoskeletal proteins and the effect of applying a membrane potential." Thesis, University of Essex, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279408.

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Schreyer, Adrian Michael. "Characterisation of protein-ligand interactions and their application to drug discovery." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609324.

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Qadir, Abdul. "The effects of endotoxaemia and omega-3 fatty acids on membrane fatty acids and cardiac G-proteins." Thesis, University of Aberdeen, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300911.

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Dietary manipulation was undertaken with the aim of influencing membrane composition and improving adrenergic dysfunction. The objective was to explore the effects of 5 days of continuous intra-duodenal feeding of diets containing n-3 PUFA on myocyte membrane phospholipids, contractility and G-protein. The control diet was omega-6 (n-6) PUFA from safflower oil. The animals were infused with either saline or endotoxin (1 mg/kg) in the last 24 hours. The diets resulted in incorporation of lipids with alteration in myocyte membrane lipid composition. The relative percentage of n-3 PUFA was increased in the fish oil group (22.61± 1.30, 20.46±1.35 for control and endotoxin) compared to safflower oil group (15.21±1.77, 14.16±0.56 for control and endotoxin). The mean inotropic response to isoprenaline was improved by feeding of n-3 PUFA enriched diets (0.175±0.027 for safflower oil endotoxin group vs 0.264±0.03 for safflower oil control, 0.261±0.064 for fish oil control and 0.275±0.073 for fish oil endotoxin group). The adenylyl cyclase activity on forskolin stimulation was not affected by diet or endotoxin (0.165±0.036, 0.176±0.058 for safflower control and endotoxin, and 0.163±0.036, 0.173±0.017 for fish oil control and endotoxin). The data on forskolin stimulation suggested distal contractile mechanisms were intact and that the defect in βAR signal was occurring at a site proximal to adenylyl cyclase. Sodium fluoride, a direct activator of G-proteins revealed a much greater degree of stimulation in the fish oil endotoxin group compared to the safflower oil endotoxin group (0.013±0.030 for safflower oil endotoxin, 0.053±0.015 for fish oil endotoxin and 0.055±0.022 for safflower oil control, 0.088±0.26 for fish oil control). The mean relative percentage of Gαo subunits was reduced in fish oil endotoxin group compared to safflower oil endotoxin group (16.65±3.01 for fish oil endotoxin, 25.37±1.29 for safflower oil endotoxin and 15.63±0.91 for safflower oil control, 13.06±2.70 for fish oil control). In summary, n-3 PUFA improve βAR transmembrane signal in endotoxaemia by favourably altering G-regulatory proteins possibly through membrane displacement of n-6 fatty acids.

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36

Chu, Shidong. "SOLID-STATE NMR SPECTROSCOPIC STUDIES OF PROTEINS AND SMALL MOLECULES IN PHOSPHOLIPID MEMBRANES." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1280860755.

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Niegowski, Damian. "Structural biology of integral membrane proteins from methods to molecular mechanisms /." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm Univeristy, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-30069.

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Illergård, Kristoffer, Simone Callegari, and Arne Elofsson. "MPRAP : An accessibility predictor for a-helical transmem-brane proteins that performs well inside and outside the membrane." Stockholms universitet, Institutionen för biokemi och biofysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-49473.

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Background: In water-soluble proteins it is energetically favorable to bury hydrophobic residues and to expose polar and charged residues. In contrast to water soluble proteins, transmembrane proteins face three distinct environments; a hydrophobic lipid environment inside the membrane, a hydrophilic water environment outside the membrane and an interface region rich in phospholipid head-groups. Therefore, it is energetically favorable for transmembrane proteins to expose different types of residues in the different regions. Results: Investigations of a set of structurally determined transmembrane proteins showed that the composition of solvent exposed residues differs significantly inside and outside the membrane. In contrast, residues buried within the interior of a protein show a much smaller difference. However, in all regions exposed residues are less conserved than buried residues. Further, we found that current state-of-the-art predictors for surface area are optimized for one of the regions and perform badly in the other regions. To circumvent this limitation we developed a new predictor, MPRAP, that performs well in all regions. In addition, MPRAP performs better on complete membrane proteins than a combination of specialized predictors and acceptably on water-soluble proteins. A web-server of MPRAP is available at http://mprap.cbr.su.se/ Conclusion: By including complete a-helical transmembrane proteins in the training MPRAP is able to predict surface accessibility accurately both inside and outside the membrane. This predictor can aid in the prediction of 3D-structure, and in the identification of erroneous protein structures.

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George, Kimberly Suzanne. "The Roles of Membrane Rafts in Ultraviolet Light-Induced Association of Apoptotic Proteins." Ohio University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1320893267.

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40

Banna, Christopher David. "Characterization of DAP1/YPL170W [electronic resource] : the saccharomyces cerevisiae membrane associated progesterone receptor (MAPR)homologue." Available online, Georgia Institute of Technology, 2005, 2004. http://etd.gatech.edu/theses/available/etd-01072005-125512/unrestricted/banna%5Fchristopher%5Fd%5F200505%5Fphd.pdf.

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Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2005.
Choi, Jung, Committee Chair ; Tornabene, Thomas, Committee Member ; Chernoff, Yuri, Committee Member ; Hall, Dwight, Committee Member ; Doyle, Donald, Committee Member. Includes bibliographical references.

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41

Soman, Raunak Jay. "Structure-function studies and polarity and charge as substrate determinants for the E. coli YidC." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405507449.

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42

Mitakidis, Nikolaos. "Structural studies of cell surface signalling molecules for neuronal guidance and connectivity." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:67a41765-afb6-4cbe-ae60-884773127b6c.

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Signal transduction is critical during the lifetime of a neuron as it navigates to reach its targets, forms functional synaptic connections and adjusts the molecular architecture of these connections in an activity-dependent manner. Understanding the molecular organisation of components required for neuronal signalling will provide novel biological insight and can contribute to the design of therapeutics for neurodevelopmental and neurodegenerative disorders. The focus of the thesis is on determining mechanistic molecular details of a number of distinct cell surface systems implicated in neuronal signalling. Crystallographic studies on the cell surface complex between Eph receptor A4 and ephrinA5 contributed to understanding how the modes of higher order arrangements of receptors involved in guidance affect signal transduction across the membrane. A set of structural and biophysical studies addressed the proteoglycan regulation of RPTPσ-TrkCtrans-synaptic interaction and contributed to deciphering the principles of the switch from axonal growth to synapse establishment and formation. A crystallographic and biochemical analysis of the neuronal C1q-like family, enabled mapping their interactions with potential synaptic partners, and guided functional studies aimed at elucidating their roles in the maintenance of synaptic integrity. Preliminary work on the neuronal Sigma-1 receptor chaperone laid the foundations for the structural determination of this receptor.

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43

Georgiev, Alexander. "Membrane Stress and the Role of GYF Domain Proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm university, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7764.

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Howard, Alison. "Identification and molecular characterisation of two proteins, calbindin-DK9 and basolateral membrane calcium ATPase, involved in mammalian intestinal calcium absorption." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243819.

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Delman, Emily. "Effects of Synthetic Ligands onHeterodimer Pairs Regarding Full-Length Human PPARa, RXRa and LXRa." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472204976.

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Bottorf, Lauren Marie. "Developing Electron Paramagnetic Resonance Spectroscopy Methods for Secondary Structural Characterization of Membrane Proteins." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami1510164534760125.

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47

Zhu, Lu. "Studies on Substrate Determinants of YidC/Sec Pathway and Insertion/Folding of Membrane Proteins in E.Coli." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354696705.

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Hwang, William. "Droplet interface bilayers for the study of membrane proteins." Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:0ba680ba-75f1-4cd9-9600-3e251b948a3d.

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Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. The discovery and characterization of an engineered nanopore with diode-like properties is enabling the construction of DIB networks capable of biochemical computing. Moreover, DIB networks might be used as model systems for the study of membrane-based biological phenomena. We develop and experimentally validate an electrical modeling approach for DIB networks. Electrical circuit simulations will be important in guiding the development of increasingly complex DIB networks. In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. Towards this end, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both α-helical and β-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores. Even with the small volumes (~100 nL) that can be used to form DIBs, the separation between two adjacent bilayers in a DIB network is typically still hundreds of microns. In contrast, dual-membrane spanning proteins require the bilayer separation to be much smaller; for example, the bilayer separation for gap junctions must be less than 5 nm. We designed a double bilayer system that consists of two monolayer-coated aqueous spheres brought into contact with each side of a water film submerged in an oil-lipid solution. The spheres could be brought close enough together such that they physically deflected without rupturing the double bilayer. Future work on quantifying the bilayer separation and studying dual-membrane spanning proteins with the double bilayer platform is planned.

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Misquitta, Yohann Reynold. "The rational design of monoacylglycerols for use as matrices for the crystallization of membrane proteins." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141940412.

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Schou, Magnus. "Synthesis and evaluation of new PET radioligands for imaging central norepinephrine transporters /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-773-1/.

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Dissertations / Theses on the topic 'Ligands (Biochemistry) Membrane proteins'

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