Dissertations / Theses: 'Membrane proteins; Biophysics'

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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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Ranatunga, Kishani M. "Computational studies of ion channel permeation and selectivity." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325774.

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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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Sergeev, Mikhail. "Measurement of oligomerization states of membrane proteins via spatial fluorescence intensity fluctuation analysis." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96703.

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The objective of this thesis is the development and use of a novel fluorescence fluctuation technique for determining the oligomeric state of fluorescently labeled proteins in situ via microscopy imaging. High order moment analysis of fluorescence intensity fluctuations from individual confocal laser scanning microscopy (CLSM) images applied to study monomer-oligomer distributions of fluorescently labeled proteins was developed. Using computer simulations and experiments with fluorescent microspheres and organic fluorescent dyes, the detection limits and accuracy of this statistical approach were determined. A series of control experiments were carried out to support the membrane receptor oligomerization studies in this thesis. The methods were then applied to study oligomerization states in various biological systems. Epidermal growth factor receptors (EGFR) play a critical role in cell growth, proliferation and survival. The activation steps of signal transduction pathways are known to involve EGFR oligomerization. Pharmacodynamic studies of ligand-induced clustering of EGF receptors were carried out. Spatial intensity distribution analysis (SpIDA), which accurately measures monomer-dimer distributions, was used to measure the increase in EGFR dimeric population upon addition of EGF ligand. The distribution of aggregates forming in the course of EGFR internalization was evaluated using two-population moment analysis. The findings supported an existing model proposing two distinct receptor internalization pathways. The electrogenic sodium bicarbonate cotransporter NBCe1-A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. The fluorescence moment image analysis and SpIDA were applied to study the oligomeric state of NBCe1-A in cultured mammalian cells expressing various mutants of cotransporter. Spatial fluctuation analysis revealed that NBCe1-A existed on the cell membrane predominantly as a monomer and negligibly as higher order oligomers. To measure the oligomerization state of the native cotransporter, samples of rat kidney tissues were prepared and the native NBCe1-A was immunostained using fluorescently labeled primary antibody against the wild type cotransporter. The image analysis showed that NBCe1-A was present on the proximal tubule basolateral membrane in predominantly dimeric and rarely monomeric or higher order oligomeric states. Human immunodeficiency virus (HIV) diverts the cellular ESCRT machinery to promote the release of newly formed virions from host cells. The ATPase, VPS4A, acts at a late stage of ESCRT function. It provides energy for dissociation of ESCRT complexes and membrane abscision. The fluorescence moment analysis of VPS4A-eGFP images revealed the monomeric distribution of the protein in the plasma membrane outside budding sites and formation of two to five VPS4A dodecamers at the budding sites. The combination of the results of VPS4A dynamics studies together with the VPS4A burst size analysis shed light on steps in the HIV lifecycle and release.
L'objectif de cette thèse réside dans le développement et l'utilisation d'une nouvelle technique de mesure de fluctuation de fluorescence. Cette technique d'imagerie par microscopie permet de déterminer in situ l'état d'oligomérisation de protéines couplées à un fluorophore. L'analyse par mesure de moments d'ordres supérieurs d'intensité de fluctuation de fluorescence d'images obtenues à partir d'un microscope confocal à balayage laser (CLSM) a été développée afin de mesurer la distribution en monomères/oligomères de protéines marquées par fluorescence. En utilisant des simulations par ordinateur ainsi que des expériences avec des microsphères fluorescentes, les limites de détection et l'exactitude de cette approche statistique ont pu être déterminées. Une série d'expériences contrôles a été effectuée afin de valider l'étude d'état d'oligomérisation de récepteurs membranaires présentée dans cette thèse. Cette méthode a ensuite été appliquée à l'étude de l'état d'oligomérisation dans divers systèmes biologiques. Le récepteur au facteur de croissance épidermique (EGFR) joue un rôle critique dans la croissance, la prolifération et la survie cellulaire. Les étapes d'activation des voies de transduction du signal sont connues pour impliquer l'oligomérisation de l'EGFR. Des études de pharmaco-dynamique d'agglomération provoquée par liaison d'un ligand ont été conduites. La technique d'analyse de distribution spatiale d'intensité (SpIDA), qui permet de mesurer précisément la distribution de monomères/dimères a été utilisée pour mesurer l'augmentation de la population de dimères d'EGFR après liaison du ligand (EGF). La distribution des agrégats se formant au cours de l'internalisation d'EGFR a été mesurée par analyse de moments pour deux populations. Les résultats confirment une hypothèse proposant deux voies distinctes d'internalisation du récepteur. Le co-transporteur électrogénique au bicarbonate de sodium NBCe1-A joue un rôle important dans l'absorption du bicarbonate de sodium à travers la membrane baso-latérale du tubule proximal rénal. Les analyses par moments de fluorescence et SpIDA ont été appliquées pour étudier l'état d'oligomérisation de NBCe1-A dans des cellules mammifères en cultures exprimant plusieurs mutants du co-transporteur. L'analyse de fluctuation spatiale montre que NBCe1-A est présent majoritairement sous forme de monomère sur la membrane cellulaire et de façon négligeable sous forme d'oligomères d'ordres supérieurs. Afin de mesurer l'état d'oligomérisation du co-transporteur naturel, des échantillons de reins de rats ont été préparés et NBCe1-A a été marqué par immunoréaction avec des anticorps fluorescents reconnaissant le type naturel du co-transporteur. L'analyse d'image indique que NBCe1-A est présent sous forme de dimère et rarement sous forme de monomère ou d'oligomères d'ordre supérieurs sur la membrane baso-latérale des tubules proximaux. Le virus humain d'immunodéficience (VIH) détourne la machinerie cellulaire ESCRT pour promouvoir la sortie de la cellule hôte de virions nouvellement formés. L'ATPase VPS4A est impliquée à une étape avancée de la fonction ESCRT en produisant de l'énergie pour la dissociation du complexe ESCRT et l'invagination de la membrane plasmique. L'analyse du moment de fluorescence d'images de VPS4A-eGFP montre une distribution monomérique à l'extérieur des sites de bourgeonnement ainsi que la présence de deux à quatre dodécamères sur les sites de bourgeonnement. La combinaison des résultats des études de la dynamique de VPS4A ainsi que l'analyse de la taille des pics d'intensité lumineux permet de mieux comprendre le cycle de vie du VIH ainsi que son processus de relargage.

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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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Robson, Alex J. "Single particle tracking as a tool to investigate the dynamics of integrated membrane complexes in vivo." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:7769f80c-a56d-4513-9123-1d65ef8c9911.

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The last decade has seen substantial advances in single-molecule tracking methods with nano-metre level precision. A powerful tool in single-molecule tracking is fluorescence imaging. One particular application, total internal reflection microscopy, can capture biological processes at high contrast video rate imaging at the single-particle level. This thesis presents methodologically novel methods in analysing single particle tracking data. Presented here is an application of a Bayesian statistical approach that can discriminate between the different diffusive modes that appear with the presence of membrane architecture. This algorithm is denoted BARD; a Bayesian Analysis to Ranking Diffusion. These algorithms are applied to a total internal fluorescence microscopy based experimental data of a novel membrane probe in Escherichia coli. This probe is a plasmid expressed, non-native membrane integrating trans-membrane helix and thus acts as an ideal protein based probe under no specific native control. Two experiments were performed using a combination of varying helix probe size and growth temperature experiments effectively altering the transition temperature of the membrane. These data are suggestive of a passive partitioning of the helix protein into mobile and immobile domains that emerge from the underlying phase behaviour of the membrane.

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Oglęcka, Kamila. "Biophysical studies of membrane interacting peptides derived from viral and Prion proteins." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7109.

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This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.

The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.

mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.

Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.

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Raychaudhuri, Pinky. "Bilayer formation with fluorinated amphiphiles and applications in membrane protein studies." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:f8d7ec23-7b2f-4610-b7c8-395b2660464a.

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Every cell is enclosed by a membrane which gives structure to the cell and allows for the passage of nutrients and wastes into and out of the cell. Membranes are made up of amphiphilic lipid molecules, with one water-soluble end, and one hydrophobic end. Naturally occurring and synthetic membranes are made up of double-chained amphiphiles derived from hydrocarbons. Recently, a novel class of amphiphilic molecules derived from fluorocarbons have been reported. The properties of fluorinated amphiphiles are very different to that of hydrocarbon based amphiphiles. Fluorinated amphiphiles have been previously reported to be useful in the studies of membrane proteins. In this thesis, we explore some novel uses of fluorinated amphiphiles. Chapter one: Provides a comprehensive review of the properties of fluorocarbon-based amphiphiles and discusses the existing uses of fluorinated amphiphiles in biochemical and biomedical research. Chapter two: Describes some of the important materials and methods used in this thesis including a detailed description of the proteins used and the working principles behind the techniques used in the study. Chapter three: Looks at the stability of pre-formed planar lipid bilayers in the presence of fluorinated amphiphiles (F-amphiphiles), and characterizes the behaviour of alpha-haemolysin and other proteins in liposomes and planar lipid bilayers in the presence of F-amphiphiles. We found that F-amphiphiles have an inhibitory effect on the insertion of protein into lipid bilayers, and this property has been exploited to control the number of proteins in the bilayer. Chapter four: Using droplet interface bilayers, we investigate the electrical properties and behaviour of protein(s) in bilayers formed by F-amphiphiles. The results obtained with fluorinated bilayers are compared with results obtained in conventional DPhPC lipid bilayers. This is the first ever report to carry out such an investigation and it provides insights into the formation, stability and utility of fluorinated bilayers. Chapter five: In Chapter five, we explore another aspect of droplet interface bilayers: the feasibility of using droplet interface bilayers to screen for membrane protein libraries. I have chosen to focus on certain fundamental aspects of the screening process that are sufficient to establish the feasibility of the method and to act as the proof of concept. Chapter six: Summarizes all the important results in the thesis and discusses some possible future directions of this project.

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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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Cheng, Zhiliang. "Posttargeting Events in Cotranslational Translocation Through the Sec61 Complex: a Thesis." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/1.

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The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61 mutants impact different steps in the cotranslational translocation pathway. Integral membrane proteins are cotranslationally inserted into the endoplasmic reticulum via the protein translocation channel, which mediates the translocation of lumenal domains, retention of cytosolic domains and integration of transmembrane spans into the phospholipid bilayer. We analyzed the in vivo kinetics of integration of model membrane proteins in Saccharomyces cerevisiae using ubiquitin translocation assay reporters. A signal anchor sequence from a type II membrane protein gates the translocon pore less rapidly than a cleavable signal sequence from a secretory protein. Transmembrane spans and lumenal domains are exposed to the cytosol during integration of a poly topic membrane protein. The conformational changes in the translocon that permit opening of the lumenal and lateral channel gates occur less rapidly than elongation of the nascent polypeptide. Cytosolic exposure of transmembrane spans and lumenal domains poses a challenge to the fidelity of membrane protein integration.

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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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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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Anthis, Nicholas J. "Structural studies of integrin activation." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:caf0f76f-b05a-4b72-8394-5f24de3fd5df.

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Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.

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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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Hirst-Dunton, Thomas Alexander. "Using molecular simulations to parameterize discrete models of protein movement in the membrane." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:893568e9-696f-47e7-8495-59ecfb810459.

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The work presented in this thesis centres on the development of a work-flow in which coarse-grained molecular dynamics (MD) simulations of a planar phospholipid bilayer, containing membrane proteins, is used to parameterize a larger-scale simplified bilayer model. Using this work-flow, repeat simulations and simulations of larger systems are possible, better enabling the calculation of bulk statistics for the system. The larger-scale simulations can be run on commercial hardware, once the initial parameterization has been performed. In the simplified representation, each protein was initially only represented by the position of its centre of mass and later with the inclusion of its orientation. The membrane protein used throughout most of this work was the bacterial outer membrane protein NanC, a member of the KdgM family of proteins. To parameterize the motion and interaction of proteins using MD, the potential of mean force (PMF) for the pairwise association of two proteins in a bilayer was calculated for a variety of orientational combinations, using a modified umbrella sampling procedure. The relative orientations chosen represented extreme examples of the contact regimes between the two proteins: they approximately corresponded to maxima and minima of the solvent inaccessible surface area, calculated when the proteins were in contact. These PMFs showed that there was a correlation between the buried surface area and the depth of the potential well in the PMF; this is something that, to date, has only been observed in these relatively-'featureless' membrane proteins (but is seen in globular proteins), where the effect of the interactions with lipids in the bilayer plays a larger role. Features in the PMF were observed that resulted from the preferential organization of lipids in the region between the two proteins. These features were small wells in the PMF, which occurred at protein separations that corresponded to the intervening lipids being optimally packed between the proteins. This result further highlighted the role that the lipids in the bilayer played in the interaction between the NanC proteins. The simplified bilayer model was parameterized using the PMFs and the relationship between buried surface area and potential well depth. The initial model included only the proteins' positions. A series of Monte Carlo simulations were performed in order to compare the system behaviour to that of an equivalent MD simulation. Initially, the MD simulation and our parameterized model did not show a good agreement, so a Monte Carlo scheme that incorporated cluster-based movements was implemented. The agreement between the MD simulation and the simulations of our model using the cluster-based scheme, when comparing diffusive and clustering behaviour, was good. Including the orientation-dependent features of the parameterization resulted in the emergence of behaviour that was not clearly detectable in the MD simulation. Finally, attempts were made to parameterize the model using PMFs for the association of rhodopsin from the literature. Rhodopsin was a much more complicated protein to represent: there was not a clear correlation between surface area and the features of the PMF, and the geometry of the interaction between two rhodopsins was more complicated. Simulations of the 'rows-of-dimers' system of rhodopsin, observed in disc membranes, was not entirely well represented by the model; for such a closely packed system, where the number of lipids is much closer to the number of proteins, the use of an implicit-lipid model meant that the effect of the reduced lipid mobility was not adequately captured. However, the model accurately captures the orientational composition of the system. Future work should be focussed on incorporating explicit representations of the lipid in the system so that the behaviour of close-packed systems are better represented.

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Parton, Daniel L. "Pushing the boundaries : molecular dynamics simulations of complex biological membranes." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:7ab91b51-a5ae-46b4-b6dc-3f0dd3f0b477.

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A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.

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Solcan, Nicolae Claudiu. "Biochemical and biophysical studies of the prokaryotic proton dependent oligopeptide transporters." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5ad900fb-a949-4bac-b69a-f585b44a8382.

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The proton dependent oligopeptide transporters (POT family) are members of the Major Facilitator Superfamily of secondary active transporter proteins. They use the transmembrane proton gradient to drive the uptake of di- and tripeptides into the cytoplasm. Members of the family are highly conserved in pro- and eukaryotic genomes, and in humans they are responsible for the oral absorption of many drug families, including -lactam antibiotics. Recently, the crystal structures of PepTSo and PepTSt, two prokaryotic homologues of the human proteins PepT1 and PepT2, captured the proteins in two distinct conformations, providing insight into the structural aspects of the transport mechanism. A protocol was designed for functional liposome reconstitution of POT proteins, and transport assays were conducted to characterise their substrate specificity, pH dependence and kinetic properties. Using site-directed mutagenesis, we identified binding site residues involved in peptide recognition and proton translocation, and distinguished between the two roles by comparing protein activity in proton- and peptide-driven conditions. We also investigated the roles of key residues in the conformational transitions that accompany the transport cycle, using data from biochemical assays, molecular dynamics simulations and modeling, as well as electron paramagnetic resonance measurements. In addition, several bacterial POT members were screened for crystallisation, in order to assess their stability and crystal diffraction quality in different detergents. Further work was performed with bacterial POT homologues YdgR and GkPOT, including binding studies using NMR spectroscopy and assaying drug transport in vivo and in vitro. Together, the data establish bacterial POTs as model systems for studying the mammalian oligopeptide transporters, and a mechanistic model for peptide transport is proposed.

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Gross, Linda C. M. "Applications of droplet interface bilayers : specific capacitance measurements and membrane protein corralling." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:0b7ffba6-b86d-499c-a93f-3b2fc46a427b.

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Droplet Interface Bilayers (DIBs) have a number of attributes that distinguish them from conventional artificial lipid bilayers. In particular, the ability to manipulate bilayers mechanically is explored in this thesis. Directed bilayer area changes are used to make precise measurements of the specific capacitance of DIBs and to control the two dimensional concentration of a membrane protein reconstituted in the bilayer. Chapter 1 provides a general introduction to the role of the lipid membrane en- vironment in the function of biological membranes and their integral proteins. An overview of model lipid bilayer systems is given. Chapter 2 introduces work carried out in this laboratory previously and illustrates the experimental setup of DIBs. Some important bilayer biophysical concepts are covered to provide the theoretical background to experiments in this and in later chapters. Results from the characterisation of DIBs are reported, and an account of the development of methods to manipulate the bilayer by mechanical means is given. Chapter 3 describes experiments that apply bilayer area manipulation in DIBs to achieve precise measurement of specific capacitance in a range of lipid systems. Chapter 4 reports results from experiments investigating the response of bilayer specific capacitance to an applied potential. Chapter 5 covers the background and experimental setup for total internal fluo- rescence microscopy experiments in DIBs and describes the expression, purification and characterisation of the bacterial β-barrel membrane protein pore α-Hemolysin. Chapter 6 describes experiments that apply the mechanical manipulation of bilayer area in DIBs to the corralling and control of the surface density of α-Hemolysin.

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Trueman, Steven F. "Insights Into ER Translocation Channel Gating. Structural Regulation of the Transition Between the Closed and Open Channel Conformations: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/576.

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The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7 and TM8 in Sec61p) to expose the signal sequence-binding (SSB) site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles that reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity. In the opening of the translocation channel, both the lateral and lumenal gate must open in a coordinated fashion for efficient protein translocation to occur. The lumenal gate is composed of a short helix of the loop preceding the second TM span, referred to as the plug helix, and six hydrophobic pore ring residues which form the constriction ring in the center of the channel. We identified three lateral gate polar residues and three hydrophobic residues from the plug domain that affect channel gating. Mutagenesis of the lateral gate polar cluster residues yields either a gain of function (prl phenotype) or a loss of function (translocation defect) phenotype. The combination of polar cluster mutations with each other or with plug domain mutations which cause a prl phenotype resulted in the mutually suppressive or additive phenotypes in double mutant strains. Cooperation between these residues is made possible through a structural link which connects the two translocation channel gates at their interface. The structural link provides a mechanism for the channel to coordinate the movement of multiple domains in the channel gating conformational change. Translocation assays demonstrated that this mechanism of gating regulation is particularly important for efficient protein translocation of substrates using the posttranslational translocation pathway. Our results indicate that residues from the plug and lateral gate domain form a regulatory cluster of residues responsible for efficient translocation channel gating.

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Gibson, Kaylee Roy. "STRUCTURAL AND TOPOLOGICAL CHARACTERIZATION OF KCNE1 ELUCIDATED BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPYKCNE1." Miami University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=miami1368099139.

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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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Clay, Jordan Elliott. "Molecular dissection of ionotropic glutamate receptor delta-family interactions with trans-synaptic proteins." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:07101578-6cc8-4ba6-99aa-e2a93b49d89d.

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Correct functioning of the brain relies upon the precise connectivity between the billions of neurons that make up this crucial organ. Aberrations in the formation of these elaborate neural networks lead to neurodegenerative and neuropsychiatric disorders. A synapse-spanning molecular triad, involving members of the Neurexin, Cbln and ionotropic glutamate receptor delta families of proteins, is crucial for the accurate formation and proper function of synapses in the cerebellum. This trans-synaptic complex has been implicated in the molecular mechanisms behind motor control and motor learning, and furthermore individual members have been linked to diseases such as Alzheimer’s, autism spectrum disorders and schizophrenia. The major findings presented in this thesis include: crystal structures of the amino-terminal domains (ATD) of the two members of the ionotropic glutamate receptor delta (iGluR-Delta) family, functional characterisation of the effects of disrupting the ATD interface in one member of the iGluR-Delta family, a crystal structure of the C1q domain of Cbln1, biophysical analysis of the molecular interactions within the Neurexin-Cbln1-GluD2 trans-synaptic complex, as well as evidence for the domain arrangement of the ecto-domain of the iGluR-Delta proteins. Together, these data enhance our knowledge of the molecular details of this macro-molecular complex and provide evidence to support models for the mechanisms of their involvement in synapse formation and function, thereby making a contribution to the vast and medically relevant field of molecular neurobiology.

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Ghimire, Harishchandra. "Structure, Dynamics, and Distance Measurements in Membrane Proteins and Peptides using EPR Spectroscopic Techniques." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1291739688.

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Burridge, Kevin Michael. "Application and characterization of polymer-protein and polymer-membrane interactions." Miami University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1624882451668094.

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Viveca, Lindahl. "Optimizing sampling of important events in complex biomolecular systems." Doctoral thesis, KTH, Fysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-217837.

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Proteins and DNA are large, complex molecules that carry out biological functions essential to all life. Their successful operation relies on adopting specific structures, stabilized by intra-molecular interactions between atoms. The spatial and temporal resolution required to study the mechanics of these molecules in full detail can only be obtained using computer simulations of molecular models. In a molecular dynamics simulation, a trajectory of the system is generated, which allows mapping out the states and dynamics of the molecule. However, the time and length scales characteristic of biological events are many orders of magnitude larger than the resolution needed to accurately describe the microscopic processes of the atoms. To overcome this problem, sampling methods have been developed that enhance the occurrence of rare but important events, which improves the statistics of simulation data. This thesis summarizes my work on developing the AWH method, an algorithm that adaptively optimizes sampling toward a target function and simultaneously finds and assigns probabilities to states of the simulated system. I have adapted AWH for use in molecular dynamics simulations. In doing so, I investigated the convergence of the method as a function of its input parameters and improved the robustness of the method. I have also worked on a generally applicable approach for calculating the target function in an automatic and non-arbitrary way. Traditionally, the target is set in an ad hoc way, while now sampling can be improved by 50% or more without extra effort. I have also used AWH to improve sampling in two biologically relevant applications. In one paper, we study the opening of a DNA base pair, which due to the stability of the DNA double helix only very rarely occurs spontaneously. We show that the probability of opening depends on both nearest-neighbor and longer-range sequence effect and furthermore structurally characterize the open states. In the second application the permeability and ammonia selectivity of the membrane protein aquaporin is investigated and we show that these functions are sensitive to specific mutations.

QC 20171117

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Simon, Kailene S. "Structural and Biochemical Studies of Membrane Proteins CFTR and GLUT1 Yield New Insights into the Molecular Basis of Cystic Fibrosis and Biology of Glucose Transport." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1040.

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Integral membrane proteins (IMPs) assume critical roles in cell biology and are key targets for drug discovery. Given their involvement in a wide range of diseases, the structural and functional characterization of IMPs are of significant importance. However, this remains notoriously challenging due to the difficulties of stably purifying membrane-bound, hydrophobic proteins. Compounding this, many diseases are caused by IMP mutations that further decrease their stability. One such example is cystic fibrosis (CF), which is caused by misfolding or dysfunction of the epithelial cell chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Roughly 70% of CF patients world-wide harbor the ΔF508-CFTR mutation, which interrupts CFTR’s folding, maturation, trafficking and function. No existing treatment sufficiently addresses the consequences of ΔF508, and the substantial instability that results from this mutation limits our ability to study ΔF508-CFTR in search of better treatments. To that end, my colleagues at Sanofi generated homology models of full-length wild-type and ΔF508-CFTR +/- second-site suppressor mutations (SSSMs) V510D and R1070W, and performed molecular dynamics (MD) simulations for each model. Using information obtained from this analysis, I tested several hypotheses on the mechanism by which ΔF508 destabilizes full-length CFTR and how SSSMs suppress this effect. Leveraging studies of the purified NBD1 subdomain and of full-length CFTR in a cellular context, I confirmed the prediction of a key salt-bridge interaction between V510D and K564 important to second-site suppression. Furthermore, I identified a novel class of SSSMs that support a key prediction from these analyses: that helical unraveling of TM10, within CFTR’s second transmembrane domain, is an important contributor to ΔF508-induced instability. In addition, I developed a detergent-free CFTR purification method using styrene-maleic acid (SMA) copolymer to extract the channel directly from its cell membrane along with the surrounding lipid content. The resulting particles were stable, monodisperse discs containing a single molecule of highly-purified CFTR. With this material, I optimized grid preparation techniques and carried out cryo-EM structural analysis of WT-hCFTR which resulted in 2D particle class averages which were consistent with an ABC transporter shape characteristic of CFTR, and a preliminary 3D reconstruction. This result establishes a foundation for future characterization of ΔF508-CFTR in its native state. I have also applied this SMA-based purification method to the facilitated glucose transporter GLUT1 (SLC2A1). SLC2A1 mutations contribute to a rare and developmentally debilitating disease called GLUT1-deficiency syndrome. Using SMA, I successfully extracted GLUT1 in its native state. With the application of this method, I was able to purify endogenous GLUT1 from erythrocytes, in complex with several associated proteins as well as the surrounding lipids, in its monomeric, dimeric and tetrameric forms without the use of cross-linking or chimeric mutations. These results point to the potential for studying isolated IMPs without the use of destabilizing detergents and thereby offer a pathway to analysis of wild-type and mutant membrane protein structure, function and pharmacodynamics.

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Lumb, Craig Nicholas. "Computational studies of signalling at the cell membrane." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:d5b2db00-1050-4191-8eff-3521a4885a0c.

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In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.

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

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

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Sadler, Emma Elizabeth. "Single-molecule fluorescence studies of KirBac1.1." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:214fcd74-7384-4ade-ac17-7cac5c44a05c.

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Inwardly rectifying potassium (Kir) channels are essential for controlling the excitability of eukaryotic cells, forming a key part of the inter-cellular signalling system in multi-cellular organisms. However, as prokaryotic (KirBac) channels are less technically challenging to study in vitro and have been shown to be directly homologous to eukaryotic channels, they are often studied in lieu of their mammalian counterparts. A vital feature of Kir and KirBac channels is their mechanism for opening and closing, or their gating: this study predominantly features observations of open and/or closed channel populations. A well-characterised member of the KirBac family, KirBac1.1, has been successfully expressed, purified into detergent micelles, and doubly labelled with fluorescent maleimide dyes in order to enable observation of confocal-in-solution Förster Resonance Energy Transfer (FRET) at the single molecule level. Results demonstrate single-molecule FRET signals from KirBac1.1 and therefore represent the first single-molecule FRET observations from a KirBac channel. Perturbation of the open-closed dynamic equilibrium was performed via activatory point mutations, changes in pH, and ligand binding. A protocol for reconstitution into nanodiscs was optimised in order to more closely approximate native conditions, and the single-molecule FRET observations repeated. This thesis presents a comparison between measurements made using the detergent solubilisation system and those made using nanodiscs.

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Zubcevic, Lejla. "Structure and function of bacterial ion channels." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:4585a56f-f6cd-44cb-845f-a3ac397fcf38.

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KirBac channels are prokaryotic homologs of eukaryotic inwardly-rectifying potassium channels, which have served as models for gaining insight into the structure of eukaryotic channels. This thesis focuses on the structure-function relationship in these channels. The first part of this study concerns a novel KirBac channel, KirBac9.2, which contains a unique amino acid sequence in the place of the canonical GYG selectivity filter. Although expressed and purified in a stable and functional form, the protein did not form well-diffracting crystals. Functional studies suggest that KirBac9.2 is non-selective for monovalent cations and a random mutagenesis screen identified a number of activatory mutants in the cytoplasmic domains of the channel. A full electrophysiological investigation of KirBac9.2 channel function is beyond the scope of this study. However, initial studies suggest that it is possible to record currents from KirBac9.2 channels reconstituted into lipid bilayers. The second part of this thesis investigates KirBac3.1, which is a classical KirBac channel containing the consensus GYG sequence for potassium selectivity. Five high resolution structures of a mutant channel are reported, which suggest a new feature in the gating mechanism of KirBac3.1 where a rotation of the cytoplasmic domains is linked to a change in the electrostatic environment of the cytoplasmic cavity. In addition, a functional study of the KirBac3.1 showed that the channel is highly pH sensitive.

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Blodgett, David M. "Human Erythrocyte Glucose Transporter (GLUT1) Structure, Function, and Regulation: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/326.

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The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.

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Brady, Jacob Peter. "The molecular basis for ER tubule formation." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:fb5ce78d-0bc8-46dd-9552-04f1f1ec1d0f.

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Integral membrane proteins of the DP1 and reticulon families are responsible for maintaining the high membrane curvature required for both smooth ER tubules and the edges of ER sheets. Mutations in these proteins lead to motor neurone diseases such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain Reticulon Homology Domains (RHD) that have unusually long (≈30 aa) hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilise membrane curvature. I have uncovered the secondary structure and dynamics of the DP1 protein Yop1p and identified a C-terminal conserved amphipathic helix that on its own interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming amphipathic helices. Together, these results indicate that amphipathic helices play a previously unrecognised role in RHD membrane curvature stabilisation. This work paves the way towards full structure determination of Yop1p by solution state NMR and marks the first high structural resolution study on an RHD protein.

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Campos, Gustavo Scanavachi Moreira. "Bioestimulação da proteína de membrana Na,K-ATPase por laser de baixa intensidade: atividade e propriedades estruturais." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/43/43134/tde-27012015-101128/.

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A Na, K-ATPase é uma proteína que realiza o transporte ativo de cátions, se encontra na membrana plasmática de praticamente todas células animais e é formada por três subunidades: (110 kDa), (50 kDa) e (10 kDa). Neste trabalho, realizou-se a extração da proteína Na,K-ATPase de rim de coelho que foi preparada em 3 diferentes condições (i) fração de membrana rica em Na,K-ATPase; (ii) solubilizada e purificada em C12E8 e (iii) reconstituída em DPPC: DPPE lipossomo (1:1 lipídio:lipídio, 1:3 lipídio:proteína). Através de medidas de Espalhamento de Luz Dinâmico (DLS), Espectroscopia de Absorção (ABS) e Espalhamento de Raio-X a Baixos Ângulos (SAXS), associadas à medidas de atividade enzimática, constatou-se que a amostra de Na,K-ATPase solubilizada e purificada em C12E8 é constituida por diferentes agregados/oligômeros em solução. Com o intuito de eliminar os grandes agregados/oligômeros da amostra realizou-se a filtração (poro de 220 nm) e a adição do surfactante dodecil sulfato de sódio (SDS) e ambos procedimentos foram capazes de eliminar as populações de grandes agregados e/ou grandes oligômeros. A retirada destas populações pelo filtro promoveu um aumento de atividade específica da enzima. Já o SDS deve promover alterações conformacionais na estrutura da proteína que causam a inativação da mesma. Investigou-se variações de atividade da Na, K-ATPase através da irradiação da proteína presente em fração de membrana e reconstituída em lipossomo por meio de três lasers de baixa intensidade com comprimentos de onda diferentes: = 532 nm (5 mW), = 650 nm (50 mW) e = 780 nm (50 mW). Demonstrou-se que a variação da atividade enzimática depende do valor de dose de energia depositada, independe do comprimento de onda estudado neste intervalo e retorna para o nível basal após 6 horas.
The Na, K-ATPase is an active cation transporter protein, which is found in the plasma membrane of virtually all animal cells and it is comprised of three subunits: (110 kDa), (50 kDa) and (10 kDa). In this work, we performed the extraction of protein Na, K-ATPase from the kidney of adult rabbit for three different enzyme preparations (i) membrane-bound fraction; (ii) C12E8 solubilized and purified and (iii) reconstituted in DPPC: DPPE liposome (1: 1 - lipid: lipid, 1:3 - lipid:protein). Dynamic Light Scattering (DLS), Absorption Spectroscopy (ABS) and Small Angle X-ray Scattering (SAXS) were employed, associated with enzyme activity measurements. The results revealed that Na, K-ATPase C12E8-solubilized and purified is composed by different aggregates/oligomers. With the aim of eliminating large aggregates/oligomers from the protein sample, filtration (pore size 220 nm) and surfactant sodium dodecyl sulfate (SDS) addition were used. Both procedures were able to eliminate populations composed of large aggregates and/or large oligomers. The removal of these populations by the filter promoted an increase in the specific activity of the enzyme. On the other hand, SDS must promote conformational changes in the protein structure that inactivate thereof. Finally, here we also investigated variations of Na, K-ATPase activity present in the membrane-bound fraction and reconstituted in liposome under irradiation of three low-intensity lasers with different wavelengths: = 532 nm (5mW), = 650 nm (50 mW) and = 780 nm (50 mW). The results give support to the conclusion that the change in the enzymatic activity depends upon the amount of energy dose deposited, it is independent of the wavelength in the studied range and returns to the basal level after 6 hours.

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Villar, Gabriel. "Aqueous droplet networks for functional tissue-like materials." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:602f9161-368c-48c0-9619-7974f743f2f2.

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An aqueous droplet in a solution of lipids in oil acquires a lipid monolayer coat, and two such droplets adhere to form a bilayer at their interface. Networks of droplets have been constructed in this way that function as light sensors, batteries and electrical circuits by using membrane proteins incorporated into the bilayers. However, the droplets have been confined to a bulk oil phase, which precludes direct communication with physiological environments. Further, the networks typically have been assembled manually, which limits their scale and complexity. This thesis addresses these limitations, and thereby enables prospective medical and technological applications for droplet networks. In the first part of the work, defined droplet networks are encapsulated within mm-scale drops of oil in water to form structures called multisomes. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents of the droplets can be released by changing the pH or temperature of the surrounding solution. Multisomes have potential applications in synthetic biology and medicine. In the second part of the work, a three-dimensional printing technique is developed that allows the construction of complex networks of tens of thousands of heterologous droplets ~50 µm in diameter. The droplets form a self-supporting material in bulk oil or water analogous to biological tissue. The mechanical properties of the material are calculated to be similar to those of soft tissues. Membrane proteins can be printed in specific droplets, for example to establish a conductive pathway through an otherwise insulating network. Further, the networks can be programmed by osmolarity gradients to fold into designed shapes. Printed droplet networks can serve as platforms for soft devices, and might be interfaced with living tissues for medical applications.

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Kurauskas, Vilius. "Fonction d'une protéine membranaire : étude structurale et dynamique par RMN." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV005/document.

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L’utilisation de détergents est inévitable pour les études structurales des protéines membranaires. Dodecylphosphocholine (DPC) est un des détergents les plus utilisés pour ce type d’études employant la spectroscopie de résonance magnétique nucléaire (RMN) en solution. L’effet des détergents sur la structure et la dynamique des macromolécules est une problématique importante, mais peu étudiée à ce jour. Dans cette étude nous avons caractérisé la dynamique à l’échelle de la milliseconde, la liaison des substrats ainsi que des propriétés structurales de trois protéines membranaires différentes solubilisées dans des micelles de DPC. Ces protéines font partie de la famille des transporteurs mitochondriaux et nous avons choisi les séquences de la levure (ORC1, GGC1, AAC3). Nous avons détecté de la dynamique à l’échelle de la milliseconde qui est distribuée d’une manière asymétrique à travers la structure. En contradiction avec des propos de la littérature, nous montrons que cette dynamique n’est pas corrélée à la fonction, puisqu’elle n’est pas modifiée par des mutations qui inhibent le transport effectué par ces protéines quand elles sont reconstituées dans des liposomes. En plus, nous avons pu montrer que leur spécificité par rapport aux substrats, n’est pas conservée quand ces transporteurs sont reconstitués dans du DPC, mettant en question leur fonctionnalité dans ce détergent. La RMN a aussi permis de démontrer que les structures tertiaire et secondaire sont perturbées dans les micelles avec quelques hélices transmembranaires apparaissant exposées au solvant. Nous avons donc conclu que la présence du détergent a un effet fort sur les trois transporteurs mitochondriaux de notre étude et probablement d’autres protéines similaires, en les rendant très flexible. Nos résultats indiquent un probable effet général de ce détergent sur les protéines membranaires, comme nous le discutons dans une analyse détaillée de quelques études de protéines membranaires décrites dans la littérature. Dans la seconde partie de ce travail, nous avons adressé une question fondamentale de la dynamique des protéines: comment se comportent les protéines dans des cristaux ? Nous avons étudié la dynamique de l’ubiquitine cristalline à l’échelle de la milliseconde afin de comprendre l’influence de la maille cristalline sur ce type de mouvement. Pour ce faire, nous avons employé la RMN à l’état solide et des simulations de dynamique moléculaire de la protéine dans différents réseaux cristallins distincts. Il est intéressant à noter que dans ces cristaux on détecte toujours des processus locaux d’échange dynamique sur une échelle de temps de la milliseconde. Cependant, en comparant les résultats obtenus avec différentes formes cristallines, nous constatons que les paramètres thermodynamiques des différents états en échange et les vitesses d’interconversion entre ces dernières sont significativement modifiés par les contacts cristallins. De plus, nous avons détecté des mouvements globaux de type «rocking» des ces molécules à l’état cristallin qui surviennent également à l’échelle de la milliseconde. Ceci suggère que les mouvements globaux et locaux sont corrélés. Cette observation ouvre la discussion de l’importance de ce type de mouvements pour la qualité et l’interprétation des données des expériences de diffraction des rayons-X
The use of detergents is often unavoidable in the structural studies of membrane proteins. Dodecylphosphocholine (DPC) is one of the most commonly used detergents for such studies in solution state NMR spectroscopy. The effect of detergent on structure and dynamics remains an important and poorly understood question. In this study we have investigated millisecond dynamics, substrate binding and structural features of three different yeast proteins from mitochondrial carrier family (GGC1, ORC1 and AAC3) in DPC micelles. We have detected millisecond dynamics, which are asymmetrically distributed across the structure. Contrary to previous claims, we show that these dynamics are unrelated to function, as they are not affected by the substitutions which abolish mitochondrial carrier transport in proteoliposomes. Furthermore, we could show that the very well-defined substrate specificity of these proteins in membranes is abolished when they are reconstituted in DPC, questioning their functionality. Structural investigations have revealed that both tertiary and secondary structures of these carriers are perturbed in DPC micelles, with some TM helices showing substantial solvent exposure. We have concluded from these observations that DPC detergent strongly perturbs these, and likely other mitochondrial carriers by rendering them very flexible. Our findings point to a possibly general effect of this detergent on membrane proteins, as we discuss with examples of previously studied membrane proteins. In the second part we have addressed a fundamental question of protein dynamics: how do proteins move inside crystals? We have investigated ms dynamics in a crystalline ubiquitin to gain the insight on the impact of the crystalline lattice on such motions, using solid-state NMR and ms long MD simulations of explicit crystal arrangements. Interestingly a local dynamic exchange process on a ms time scale is still present in crystals. However, by comparing different crystal forms we establish that the thermodynamics of the exchanging states and their interconversion rate constants are significantly altered by the crystal contacts. Furthermore, we detect overall "rocking" motion of molecules in the crystal, occurring on a tens-of-ms time scale, and provide evidence that overall and local motion are coupled. We discuss the implications of ms dynamics on the data quality in X-ray diffraction experiments

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37

Tengel, Tobias. "Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1472.

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Shanley, Samantha Jane. "A glycopore for bacterial sensing." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:06fe9bce-6bf2-4f61-b4d8-014cb9df3fc0.

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Increasing antibiotic resistance has created a need to develop rapid and reliable methods to identify bacteria and provide pertinent information to ensure suitable antibiotics or sugar therapeutics can be chosen for treatment. Carbohydrate structures attached to proteins on host cell surfaces provide a binding point for many pathogens, including bacteria. These structures can be mimicked using single monosaccharides glycosylated to alpha-hemolysin (alpha-HL). Alpha-HL is a beta-barrel pore-forming toxin secreted by Staphylococcus aureus that forms an SDS stable heptamer, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified heptamers can be reconstituted into planar lipid bilayers and studied at the single channel level. Through single channel recordings the effects of sugar-linker lengths, different glycans and the interaction between the ‘Glycopore’ and sugar binding molecules can be studied. The glycopore, therefore, acts as a scaffold for analysing protein-sugar interactions. Studies in this thesis have focused on the synthesis of carbohydrates for site-selective protein glycosylation; cloning and in vitro transcription translation of alpha-HL monomers; and glycosylation and oligomerisation of alpha-HL to form glycopores suitable for lectin-binding studies. Lectins DC-SIGN and FimH have been expressed in Escherichia coli and these lectins as well as others have been screened using alpha-HL glycopores. The glycopores have also been investigated with bacteria in serum in a controlled molecule-specific manner using single-channel electrical recording. In this work glycosylated alpha-HL-monomers have been found to form stable heptamers which can be formed by oligomerisation on red blood cell membranes. The purified glycopores were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings an optimised glycopore has been shown to be effective in distinguishing lectins alone and in a mixture and has afforded qualitative and quantitative information about the binding interactions between carbohydrates and sugar binding proteins. Furthermore, the glycopore has been used to sense bacteria which may provide an insight into modes of bacterial infection. In addition, a multivalent glycopore has been formed which has proved preliminary information about the effects of multivalency in lectin binding. The design and synthesis of non-beta-lactam antibiotic candidates and their evaluation has also been carried out.

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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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Savage, David F. "Towards membrane protein structure determination." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3251931.

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Yin, Daniel. "Biophysical investigations into membrane-active peptides and proteins." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:25401447-e37b-4c07-a22d-29718958ac48.

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The inexorable spread of antibiotic or antimicrobial resistance is a global problem, described by the UK Chief Medical Officer to be "as big a threat as terrorism". Due to uncontrolled, unnecessary overuse of antibiotics in medicine and agriculture, bacterial resistance has evolved to even the antibiotics of last resort. Antimicrobial peptides (AMPs) are a promising class of organic molecule that have been proposed to exert a potent antimicrobial effect, which, directly or indirectly, involve complex interactions with cell membranes. Three broad mechanisms have been proposed for AMPs: carpet, barrel-stave and toroidal pore. However, the molecular basis for the mode of action of AMPs, and the relationship between primary structure and antimicrobial activity, remains poorly understood. In this thesis, interactions of membrane-active peptides and proteins with model lipid membranes are studied, to understand better the peptide-lipid interactions of two de novo AMPs and a functionally related protein puroindoline-b (pinB), which is implicated in antimicrobial plant defence. Quartz crystal microbalance (QCM), solid-state nuclear magnetic resonance (ssNMR), electron paramagnetic resonance (EPR) and neutron reflectivity (NR) are used to achieve this. The two AMPs were designed rationally with their primary structure predicted to display specific peptide-lipid interactions. Tilamin (tilted antimicrobial insert) was designed by modifying amhelin (antimicrobial insert), a pore-forming AMP. The modified peptide was predicted to disrupt model membranes mimicking bacterial membranes via a different mode of action to transmembrane barrel stave pore formation. Chom (chopped cecropin mutant) was designed by shortening the length of a natural AMP, cecropin-B, and was predicted to operate via a carpet mechanism. To model the biophysical properties such as morphology, thickness and charge of native membranes, simplified phospholipid liposomes were used to better understand the membrane-perturbing influence of the AMPs, and whether this was correlated with antimicrobial activity. In the presence of anionic model membranes (mimicking Gram-negative inner membranes and Gram-positive membranes), tilamin and chom adopt amphipathic alpha helix conformations as determined by circular dichroism, while remaining unstructured in solution and in the presence of zwitterionic model membranes mimicking mammalian model membranes. Adoption of a folded conformation appears to be important for the lytic effect of the AMPs. Calcein leakage experiments performed show that the AMPs induce leakage of calcein from the interior of anionic liposomes, consistent with the proposal that membrane permeabilisation is important for antimicrobial activity. The peptide-lipid interactions of the AMPs were then probed using QCM and ssNMR, giving mechanistic evidence that chom operates via the carpet mechanism as predicted. The nature of the mode of action of tilamin remained uncertain. From order parameters of lipids in bilayers, obtained using ssNMR upon interaction with tilamin, a toroidal pore mechanism was proposed, along with a new mode of action that caused monoleaflet poration, though it was not possible to resolve the two mechanism based on data obtained from symmetrical vesicles alone. Adapting a newly established protocol to control the leaflet distribution of lipids in model membranes, an asymmetrically distributed nitroxide probe reveals for the first time leaflet-specific peptide-lipid interactions using cw-EPR. Tilamin shows changes in bilayer lipid order parameters that do not match those seen for either an all-surface or transmembrane control peptide, indicating more complex interactions. Unique QCM data, heterogeneous changes in order parameter profiles observed with acylchain ²H ssNMR, as well as a lack of interaction with the inner leaflets of anionic model membranes seen by cw-EPR taken in combination suggest tilamin operates via a more complicated mechanism. Supported by tilt angles obtained by geometric analysis of labelled alanines (GALA) of deuterium-labelled tilamin and atomic force microscopy (AFM) imaging performed with collaborators, the results are consistent with a new mechanism; monolayer poration. The puroindolines are also studied, due to their potential role as antimicrobial proteins in food safety, and in controlling wheat endosperm texture. The mode of insertion of wild-type puroindoline-B (pinB+) and a single-point mutant (pinBs) into bacterial model bilayers was probed for the first time with ssNMR, EPR and NR. In contrast to previous work on monolayers, pinBs does not cause changes in bilayer lipid order in the gel phase, while pinB+ forms a protein layer on the surface of a membrane. The results suggest that in more native-like model membranes, the tryptophan-rich domain (TRD) of pinBs and pinB+ greatly affects the membrane binding properties, with implications for the role of the proteins in vivo.

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Turner, Christina Elizabeth. "Biophysical studies of SNARE protein-membrane interactions." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/5276.

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SNARE (Soluble NSF (N-ethylmaleimide Sensitive Fusion) Attachment Protein Receptors) proteins have been linked to the membrane fusion mechanism since 1993 as fusion proteins and have been suggested to be the minimal machinery. The complexity of the fusion process means that many questions remain unanswered as to how SNARE proteins perform their role. The most favoured model (the stalk model) does not involve proteins directly and so the influence of the SNARE proteins on lipid properties is of interest. In this thesis, work is presented which investigates how these proteins may manipulate membrane properties in order to promote fusion. Purified proteins solutions of His6-VAMP2 (Vesicle Associated Membrane Protein 2), His6-SNAP-25 (Synaptosomal-associated Protein 25) and a truncated form of Syntaxin 1A (His6-ΔN-Syx 1A, w.t aa 181-288) were obtained following bacterial over-expression. Fluorescent versions of His6-VAMP2 and His6-ΔN-Syx 1A were produced by the addition of cysteine residues to the C-terminus followed by labelling using Alexa Fluor® 488-C5-maleimide and Alexa Fluor® 555-C2-maleimide respectively. These fluorescent proteins were used to establish that the purified protein inserted into model lipid bilayers. The effect of SNARE protein incorporation on the relaxed curvature of bilayers was explored by examining giant unilamellar vesicles grown using electroformation. Bilayers containing either 1:300 His6-VAMP2: DOPC or 1:1:600 His6-SNAP-25: His6-ΔN-Syx 1A: DOPC were smaller than pure DOPC vesicles, indicating that SNARE proteins increase the relaxed curvature of the bilayer. Analysis of these vesicles by micropipette aspiration suggested that VAMP2 lowered the bending rigidity of the membrane and a reduction in the area expansion modulus relative to the pure lipid bilayer was found. The t-SNARE sample also indicated a reduction in bending rigidity but the area expansion modulus was found to increase. These latter results are thought to be due to the formation of protein aggregates. Lipid mixing assays were conducted to investigate how changes in the properties of liposome bilayers affected fusion rates. It was found that the addition of DOPE to DOPC bilayers increased the rate of hemifusion and this was also found for cholesterol addition, suggesting both components are fusogens. The rate of hemifusion rose continually upon DOPE addition but reached a plateau in the cholesterol study shortly after 10 mol%. Despite this, the fusion rates for the cholesterol study were generally higher than the same mol% DOPE added. The changes in fusion rates observed have been explained by considering the impact of the additives on the free energy and stored curvature elastic stress of membranes as well as the change in the energy of formation of intermediate structures. From the findings of this thesis it is proposed that the SNARE proteins are able to soften the membranes in which they reside. This allows the membrane to be deformed with less energy input. The strength of the SNARE complex and the force applied to the membranes during its formation increases membrane tension and reduces inter-membrane separation; promoting hemfusion. Following the action potential of the neuron it is proposed that a conformational change occurs in the synaptic SNARE complex, pulling on the hemifusion diaphragm and inducing the formation of a fusion pore.

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Holden-Dye, Kate. "Biophysical studies of photosynthetic membrane proteins from Rhodobacter sphaeroides." Thesis, University of Bristol, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499862.

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Dijkman, Patricia M. "Biophysical studies of membrane protein structure and function." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:ad0fde85-c4b6-48a1-b51b-d304aca45402.

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Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

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Botelho, Ana Vitoria. "Lipid-protein interactions: Photoreceptor membrane model." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/280765.

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G-protein coupled receptors (GPCRs) are transmembrane proteins capable of recognizing an astonishing variety of biological signals, ranging from photons of light to hormones, odorants, and neurotransmitters involved in key biological signaling processes. The aim of this work is to identify how lipid-protein interactions involving the membrane bilayer ultimately affect such vital biological functions. Here the relationship between the bilayer thickness, hydrophobic mismatch, and protein aggregation are investigated by expanding the framework of membrane-receptor interactions in terms of a new flexible surface model. Previously, we have shown how coupling of the elastic stress-strain due to mismatch of the spontaneous curvature and hydrophobic thickness at the lipid/protein interface can govern the conformational transitions of membrane proteins. This approach has now been extended to include coupling of the lateral organization of the GPCR rhodopsin to the curvature and area stress and strain of the proteolipid membrane. Rhodopsin was labeled with site-specific fluorophores, and a FRET technique was employed to probe protein association in different lipid environments. Moreover, UV-visible spectroscopy was used for thermodynamic characterization of the conformational change of rhodopsin. Lastly, the deformation of the lipids with and without rhodopsin was probed in terms of acyl chain order parameters and relaxation rates by solid-state NMR methods, giving insight into the lipid deformation. The results showed that optimal receptor activation occurs in phosphatidylcholine bilayers of 20-carbon acyl chain length, hence one can say that metarhodopsin II is likely to adopt an elongated shape. Lipids promoting aggregation, or below their gel to liquid crystalline transition temperature all favor formation of metarhodopsin I. The data also showed that association and activation of rhodopsin do not always correlate. In terms of the extended flexible surface model, the stress due to hydrophobic mismatch is coupled via the effective number of lipids surrounding the protein due to the lateral organization of the membrane. The measured changes in rhodopsin-rhodopsin interactions and membrane influences on the conformation of the protein after photoisomerization may be crucial to understanding physiological regulation of the rod disk membranes. They are relevant to understanding the complexity of biomembranes involved in many cellular mechanisms, including signal transduction.

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Bilyard, Thomas. "Single molecule studies of F1-ATPase and the application of external torque." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:9f369674-4105-4bf1-a0ce-023db1f8bd7f.

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F₁-ATPase, the sector of ATP synthase where the synthesis of cellular ATP occurs, is a rotary molecular motor in its own right. Driven by ATP hydrolysis, direct observation of the rotation of the central axis within single molecules of F₁ is possible. Operating at close to 100% efficiency, F₁ from thermophilic Bacillus has been shown to produce ~40pN˙nm of torque during rotation. This thesis details the groundwork required for the direct measurement of the torque produced by F₁ using a rotary angle clamp, an optical trapping system specifically designed for application to rotary molecular motors. Proof-of-concept experiments will be presented thereby demonstrating the ability to directly manipulate single F₁ molecules from Escherichia coli and yeast mitochondria (Saccharomyces cerevisiae), along with activation of F₁ out of its inhibited state by the application of external torque. Despite in-depth knowledge of the rotary mechanism of F₁ from thermophilic Bacillus, the rotation of F₁ from Escherichia coli is relatively poorly understood. A detailed mechanical characterization of E.coli F₁ will be presented here, with particular attention to the ground states within the catalytic cycle, notably the ATP-binding state, the catalytic state and the inhibited state. The fundamental mechanism of E.coli F₁ appears to depart little from that of F₁ from thermophilic Bacillus, although, at room temperature, chemical processes occur faster within the E.coli enzyme, in line with considerations regarding the physiological conditions of the different species. Also presented here is the verification of the rotary nature of yeast mitochondrial F₁. The torque produced by F₁ from thermophilic Bacillus, E.coli and yeast mitochondria is the same, within experimental error, despite their diverse evolutionary and environmental origins.

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Thompson, James Russell. "Imaging the assembly of the Staphylococcal pore-forming toxin alpha-Hemolysin." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:e320004a-6118-4dac-af2a-eca6e90be7ac.

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Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

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Bradshaw, Niels Raab. "Coordination of cotranslational protein targeting to the membrane." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3359542.

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Leftin, Avigdor. "Nuclear magnetic resonance probes of membrane biophysics: Structure and dynamics." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/305369.

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The phospholipid membrane is a self-assembled, dynamic molecular system that may exist alone in association with only water, or in complex systems comprised of multiple lipid types and proteins. In this dissertation the intra- and inter-molecular forces responsible for the atomistic, molecular and collective equilibrium structure and dynamics are studied by nuclear magnetic resonance spectroscopy (NMR). The multinuclear NMR measurements and various experimental techniques are able to provide data that enable the characterization of the hierarchical spatio-temporal organization of the phospholipid membrane. The experimental and theoretical studies conducted target membrane interactions ranging from model systems composed of only water and lipids, to multiple component domain forming membranes that are in association with peripheral and trans-membrane proteins. These measurements consisit of frequency spectrum lineshapes and nuclear-spin relaxation rates obtained using 2 H NMR, 13 C NMR, 31 P NMR and 1 H NMR. The changes of these experimental observables are interpreted within a statistical thermodynamic framework that allows the membrane structure, activation energies, and correlation times of motion to be determined. The cases presented demonstrate how fundamental principles of NMR spectroscopy may be applied to a host of membranes, leading to the biophysical characterization of membrane structure and dynamics.

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Reading, Eamonn. "Structural insights into membrane proteins, membrane protein-lipid interactions and drug metabolites in the gas-phase from ion mobility mass spectrometry." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f94d42d1-f870-49f9-98b5-42c9b1064e1e.

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Investigating the structures of membrane proteins and their interactions with lipids remains challenging for well-established biophysical techniques. In this thesis the use of mass spectrometry (MS) and ion mobility (IM) spectrometry were explored for the interrogation of membrane proteins, their stoichiometry, stability and interactions with lipids. The techniques used were also applied to the identification of drug metabolites. In the first two chapters reviews of both mass spectrometry methods, and membrane protein biogenesis and membrane protein-lipid interactions are presented. The first challenge for studying membrane proteins by MS was to optimise solution conditions. A detergent screening strategy was developed for this purpose (Chapter 3). The various detergent environments studied revealed dramatic differences in mass spectral quality permitting investigation of membrane protein-lipid interactions. Changes were observed in the electrospray charging of membrane proteins and trends were established from an extensive collection of membrane proteins ejected from a wide variety of detergent environments. The physicochemical principles behind the MS of membrane proteins were deduced and are presented (Chapter 4). The results of these experiments led to a deeper understanding of the ionisation processes and the influence of detergent micelles on both charge state and release mechanisms. Experiments from a range of different micelles also allowed the influence of charge and its effects on the preservation of native-like membrane protein conformations to be monitored by IM-MS. By resolving lipid-protein interactions, and by monitoring the effects of lipid binding on the unfolding of three diverse membrane protein complexes, substantial differences in the selectivity of membrane proteins for different lipids were revealed (Chapter 5). Interestingly lipids that stabilised membrane proteins in the gas-phase were found to induce modifications in structure or function thus providing an approach to assess direct lipid contributions, and to rank order lipids based on their ability to modulate membrane proteins. Using the MS approaches developed here also enabled study of the diversity of oligomeric states of the mechanosensitive channel of large conductance (MscL) (Chapter 6). Results revealed that the oligomeric state of MscL is sensitive to deletions in its C-terminal domain and to its detergent-lipid environment. Additionally, a case study with GlakoSmithKline (GSK) was undertaken using IM-MS technology but in this case applied to the identification of drug metabolites (Chapter 7). The results showed that IM-MS and molecular modelling could inform on the identity of different drug metabolites and highlights the potential of this approach in understanding the structure of various drug metabolites.

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Dissertations / Theses on the topic 'Membrane proteins; Biophysics'

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