Dissertations / Theses on the topic 'Membrane proteins and peptides'
To see the other types of publications on this topic, follow the link: Membrane proteins and peptides.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Membrane proteins and peptides.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Käll, Lukas. "Predicting transmembrane topology and signal peptides with hidden Markov models /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-719-7/.
Full text
2
Oldham, Alexis Jean. "Modulation of lipid domain formation in mixed model systems by proteins and peptides." View electronic thesis, 2008. http://dl.uncw.edu/etd/2008-1/r1/oldhama/alexisoldham.pdf.
Full text
3
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.
Full textAbstract:
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.
4
Mitchell, Stephen Anthony. "Membrane translocating peptides for the delivery of proteins." Thesis, University of Bath, 2003. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397763.
Full text
5
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.
Full textAbstract:
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.
6
Polozov, Ivan V. "Interactions of class A and class L amphipathic helical peptides with model membranes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0006/NQ30110.pdf.
Full text
7
Redeby, Theres. "Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides." Doctoral thesis, Stockholm, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-617.
Full text
8
McKinley, Laura Ellen. "Neutron reflectivity studies of bacterial membranes, peptides and proteins." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28874.
Full textAbstract:
This thesis uses neutron and x-ray reflectivity to measure the interfacial structures of three molecular components associated with bacteria. Firstly, the way in which the membrane targeting sequence of a cell division protein interacts with monolayer models for the inner leaflet of the inner membrane of bacteria was measured at the air-water interface. Secondly, the influence of lipopolysaccharide on a monolayer model for the outer leaflet of the outer membrane of Gram-negative bacteria was measured at the air-water interface, as well as how this lipopolysaccharide interacts with an antimicrobial peptide. Finally, the structure of a layer of protein found at the surface of a Gram-positive biofilm was measured at the air-water interface. Binding of the membrane targeting sequence of the MinD protein (MinD-mts) to the inner leaflet of the cytoplasmic membrane is thought to be key for bacterial cell division. Modelling this membrane as a monolayer at the air-water interface, it was found that the insertion of the MinD-mts increased with decreasing lateral pressure within the monolayer, as well as with increasing unsaturation of the lipid components, and the incorporation of cardiolipin into the monolayer. Lipopolysaccharide (LPS) is the major component of Gram-negative outer membranes, such as Escherichia coli, and can be considered as having three structural components: lipid A, a core oligosaccharide, and a variable polysaccharide chain. By incorporating LPS into a model membrane at the air-water interface, it was found that the polysaccharide chain undergoes conformational changes depending on the area per molecule. The effect of the antimicrobial peptide Pexiganan on the structure of this LPS layer was also determined, and was found to insert into the polysaccharide chain layer, but have no impact on the conformation of the chains. In nature, many bacteria live within a biofilm structure. A critical component of the Gram-positive Bacillus subtilis biofilm is a surface active amphipathic protein called BslA, which gives rise to the formation of the highly hydrophobic surface of the biofilm. The kinetics of this film formation, its thickness, and the lateral packing of the protein at the air-water interface, were measured using both neutron and x-ray reflectivity. It was found that a native BslA protein consistently formed the same structural film, whilst the structure of films formed by mutant proteins depended on the conditions under which the film was formed.
9
Whiles-Lillig, Jennifer A. "Bicelles : a new system for studying membrane associated peptides and proteins /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3022189.
Full text
10
Trifunovski, Alexandra. "On nogo signaling regulation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-906-8/.
Full text
11
Zheng, Hong. "Designing Peptides to Target Membrane Lipids and to Evaluate Fluorination of Proteins." Thesis, Boston College, 2012. http://hdl.handle.net/2345/3682.
Full textAbstract:
Thesis advisor: Jianmin GaoMy graduate research has used engineered peptides to perturb the non-covalent interactions in protein folding, protein-protein association and protein-membrane association. We have focused on understanding the fundamental principles of molecular recognition behind protein-protein and protein-membrane interactions, and further using these principles in protein engineering. This thesis includes three projects. I) Towards Small Molecule Receptors for Membrane Lipids: A Case Study on Phosphatidylserine The lipid composition and distribution of cell membranes play important roles in regulating the physiology of the cell. The lipid composition of plasma membranes is one characteristic feature that can be used to identify cell types and functions. Molecules that specifically recognize a particular lipid are useful as imaging probes for targeting cells or tissues of interest. Protein based lipid binding probes have intrinsic limitations due to their large size and poor pharmacokinetic properties such as slow clearance rate and poor in vivo stability. A plausible strategy to achieve a probe with small size and high binding affinity and selectivity is to use a peptide to mimic the protein lipid-binding domains. As a case study, a cyclic peptide that specifically targets phosphatidylserine containing membranes has been developed. This cyclic peptide is potentially capable of imaging apoptosis in vivo, and the strategy of developing this cyclic peptide can be generalized to the design of peptide-based probes for other lipid species. My research has pointed out a challenging but feasible way to design a peptide that achieves specificity and affinity similar to lipid-binding proteins. (II) Study of Apoptotic Cell Membrane (ACM) Permeant Molecules Noninvasive imaging of apoptosis is highly desirable for the diagnosis of a variety of diseases, as well as for the early prognosis of anticancer treatments. One characteristic feature of apoptotic cells that has been targeted for developing specific biomarkers is enhanced membrane permeability compared to that of healthy cells. Several unrelated molecules that are capable of selectively penetrating the apoptotic cell membrane (ACM) have recently been reported. However, the origin of the altered ACM permeability is poorly understood, as is the scope of molecular structures that can permeate through the ACM. Herein, we report a systematic investigation on the altered ACM permeability. Our results show that simple modifications of commonly used dyes (e.g. fluorescein) afford specific entry into cells at the early stages of apoptosis. The ACM appears to be permeable to molecules of various functional groups and charge, but does discriminate against molecules of large size. The new findings reported here greatly expand the pool of small molecules for imaging cell death, thus facilitating the development of noninvasive imaging agents for apoptosis. (III) Study of Aromatic-Fluorinated Aromatic Interactions in Peptide Systems Therapeutic proteins have been through a remarkable expansion in the last two decades. A general problem that they are facing is poor stability. Protein engineering focuses on solving this problem by incorporating unnatural amino acids into protein sequences to purposefully modify protein structures. Fluorinated aliphatic amino acids have been demonstrated to be effective in stabilizing protein structures and functioning as recognition motifs. In contrast, fluorinated aromatic amino acids are less studied. We investigated the effect of perturbation of fluorination on aromatic residues on the stability of protein model systems, as well as the influence on protein-protein association behavior. The results of this study provided a fundamental understanding of aromatic interactions in protein systems, and guidelines for protein engineering with fluorinated aromatics for stabilizing protein structures or directing specific protein-protein interactions
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
12
Oglęcka, Kamila. "Biophysical studies of membrane interacting peptides derived from viral and Prion proteins /." Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7109.
Full text
13
Leng, Ying. "Neuron-ligand pathfinding on surfaces modified by laminin and laminin-derived peptides." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 78 p, 2006. http://proquest.umi.com/pqdweb?did=1203562381&sid=8&Fmt=2&clientId=8331&RQT=309&VName=PQD.
Full text
14
Rigler, Per. "Investigation of peptides and membrane proteins at interfaces using Fourier transform infrared spectroscopy /." Lausanne, 2002. http://library.epfl.ch/theses/?nr=2544.
Full textAbstract:
Thèse sciences, EPF Lausanne, no 2544 (2002), Faculté SB Section de chimie. Directeur: H. Vogel ; rapporteurs: K. Gerwert, K. Johnsson, M. Textor."The results have been partially published in Sevin-Landais, A. [et al.] (2000) Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes (Biophys Chem 85, 141-152)"
15
Sengupta, Durba. "Insights into the energetics of membrane-bound peptides towards an understanding of the structural organisation of membrane proteins /." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976744465.
Full text
16
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.
Full text
17
Kaye, Elena Cortizas. "The Function of Outer Membrane Protein A (OmpA) in Yersinia pestis." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_theses/58.
Full textAbstract:
The outer membrane protein OmpA is one of the major outer membrane proteins in many species of bacteria, including the Yersiniae. Our goal was to explore the role of OmpA in Y. pestis. This encompasses the ability of Yersinia to infect and survive within macrophages, as well as to resist antimicrobial compounds. Our laboratory found that a delta ompA mutant is impaired in a macrophage-associated infectivity assay. We also found that OmpA might play a role in the ability of the bacteria to resist antimicrobial peptides, specifically polymyxin B. Aditionally, we assessed the differences in OmpA of Y. pestis and E. coli, and determined that the characteristics we have observed in Y. pestis are unique compared to what has previously been described in E. coli. Our results indicate that Y. pestis OmpA might act through known pathways of antimicrobial resistance such as the PhoPQ two-component regulatory system, although further experiments are needed to determine the precise mechanism of function OmpA. Overall, our project characterizes the different functions of OmpA in Y. pestis, both as a key player in intracellular survival and as a necessary component in conferring resistance to antimicrobial peptides.
18
Radzey, Hanna Agnes. "Synthesis of fluorescent toxin and nucleotide derivatives to specifically address membrane proteins." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://hdl.handle.net/11858/00-1735-0000-0022-6055-5.
Full text
19
Louie, Sarah. "Wnt signaling regulated by Frizzled and HIPK1 /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/6267.
Full text
20
Ismail, Vian Sdiq Ismail. "Probing lipidation of membrane active peptides and integral membrane proteins by liquid chromatography-mass spectrometry and ion mobility separation-mass spectrometry." Thesis, Durham University, 2017. http://etheses.dur.ac.uk/12424/.
Full textAbstract:
Liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS2) are shown to have the sensitivity and functionality to detect protein/peptide modifications by fatty acyl chains in vitro and in vivo studies. Further analysis was also performed by direct infusion ion mobility separation coupled with mass spectrometry (IMS-MS) or tandem mass spectrometry (IMS-MS2). Peptide lipidation in vitro was investigated using the membrane active peptide, melittin. Non-enzymatic melittin lipidation by lysophospholipids has been observed for the first time. When the effect of lysophospholipids was studied in direct competition with diacylphospholipids, the acyl transfer from the lysophospholipids is seen to be preferential with acylation visible after just 3 hour. The longer the interaction time, the greater the amount and number of modifications with double and triple acylation observed after 96 hour. The locations of the modifications identified through LC-MS2 were assigned on different sites of the peptide, including N-terminus, K7, S18, K21, K23, R22 and R24 and with the highest reactivity towards N-terminus and K23. Comparing the lipidation of synthetic melittin (SynM) with the lipidation of naturally occurring melittin from venom of honey bee (BVM) highlights the effect of the PLA2 enzyme that is naturally present in BVM. Here, the action of the enzyme to hydrolyse the diacylphospholipid at the sn-2 position to give the corresponding lysophospholipid is reflected in the acyl transfer to the BVM such that the resulting lysophospholipid clearly dominates the acyl transfer to BVM. In contrast, the acyl transfer to SynM clearly demonstrates that acyl transfer is possible in the absence of an enzyme. In vivo protein lipidation of one of the most abundant integral membrane proteins in mammalian eye lens, AQP0, was also studied. A wide range of acyl groups are shown to modify this protein at the known modification sites, N-terminus and at the amino acid residue K238, many of which are reported here for the first time. These acyl group modifications reflect the biological lipid composition of the membrane leaflet that the acylation sites are proximal to. In an attempt to further distinguish between different forms of lipidated melittin, whether with the same acyl chain modification to different amino acids or to discriminate between palmitoylation and oleoylation modifications, travelling wave ion mobility spectrometry (TWIMS) coupled with MS or MS2 was applied. Results suggested that resolving positional isomers of diacylphospholipids and lysophospholipids (sn-1 vs sn-2 positions) is not possible under the conditions described herein. However, the presence of fatty acyl chains covalently bound to melittin change the conformation of acylated melittin in the gas-phase such that for lower charge states it is possible to suggest a small degree of separation between palmitoylated and oleoylated melittin or their isomers including acylation on N-terminus vs K23. This small degree of separation is enough so that when combined with tandem mass spectrometry, the time-aligned product ion spectra are clearer and improve characterisation. To conclude, the research in this thesis has shown that two of the most abundant biomolecules, lipid and peptides/proteins that are known to exist in close proximity to each other, or interact with each other, are not as chemically inert as previously thought. This reactivity has been reflected herein via aminolysis reaction between membrane lipid composition and each of membrane active peptide, melittin and integral membrane protein, AQP0.
21
Takahashi, Satoe. "Plasma Membrane Localization of Signaling Proteins in Yeast: a Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/364.
Full textAbstract:
In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway.
Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex.
At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating.
Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane.
22
Bajaj, Vikram Singh. "Dynamic nuclear polarization in biomolecular solid state NMR : methods and applications in peptides and membrane proteins." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40874.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007.Includes bibliographical references.
Solid state NMR can probe structure and dynamics on length scales from the atomic to the supramolecular. However, low sensitivity limits its application in macromolecules. NMR sensitivity can be improved by dynamic nuclear polarization (DNP), in which electron polarization is transferred to nuclei. We present applications of magic angle spinning NMR that demonstrate its utility for the determination of structure at atomic resolution. We then present new techniques and instrumentation for DNP that permit these methods to be applied to larger systems such as membrane proteins. These applications rest on several advances in instrumentation: millimeter-wave sources and conduits of power to the sample; low-temperature MAS probes incorporating millimeter-wave transmission; cryogenics and pneumatic control systems. We describe a 380 MHz DNP spectrometer incorporating a 250 GHz gyrotron oscillator and present the theory and operation of a 460 GHz gyrotron at the second harmonic of electron cyclotron resonance. We have applied DNP to study trapped photo cycle intermediates of the archael membrane protein bacteriorhodopsin, a light-driven transmembrane ion pump.
(cont.) We have observed the K photointermediate for the first time by NMR and found unexpected conformational heterogeneity in the L intermediate. With multidimensional correlation spectroscopy, we have assigned active site resonances in conformational mixtures of photointermediates of [U-13C,'SN]-bR with high sensitivity. By using non-linear sampling of indirect dimensions, we have observed transient product of K accumulation. We present frequency-selective experiments for amino acid-selective assignments and the measurement of heteronuclear distances and torsion angles in [U-13C, N]-bR and discuss the relevance of these results to its photocycle. In addition, we describe several applications of solid state NMR, including a study of dynamic and structural phase transitions in peptides and proteins near the canonical glass transition temperature. We present resonance width experiments that can be used to measure homonuclear and heteronuclear dipolar couplings in uniformly labeled solids.
(cont.) Finally, we discuss applications to amyloid fibrils, which are protein aggregates that are implicated in diseases of protein misfolding. We report the atomic resolution structure of the disease-associated L 111M mutant of TTR105-115 in an amyloid fibril, and information about the supramolecular structure of fibrils from WT TTRos05115.
by Vikram Singh Bajaj.
Ph.D.
23
Vermeer, Louic Sebichniev. "NMR struture determination and MD simultations of membrane peptides and proteins : a peptide derived from H+-V-ATPase subunit alpha, and MscL." Toulouse 3, 2009. http://thesesups.ups-tlse.fr/492/.
Full textAbstract:
H+-V-ATPase est une protéine responsable de la translocation des protons. Les propriétés structurales d'un peptide dérivé de la sous-unité a de H+-V-ATPase, ont été étudiées. La structure dans des micelles de SDS a été déterminée par RMN. Il a été démontré par dichroisme circulaire que le peptide est structuré en feuillet beta dans l'octylglucoside à haut pH, tandis qu'il présente 60\% d'hélice alpha dans le SDS. Ces observations ont été expliquées par des simulations de dynamique moléculaire, qui ont démontré que le peptide est inseré dans la micelle de SDS à haut pH, alors qu'en présence d'octylglucoside il est en interaction avec la surface de la micelle. La structure RMN en combinaison avec les simulations nous a permis d'identifier les residues participant au canal périplasmique. Dans la deuxième partie de la thèse, des essais de préparation d'échantillons du canal mécanosensible de haute conductance (MscL) dans des bicouches orientées de lipides ont été réalisés afin d'étudier cette protéine par RMN du solideThe structural properties of a peptide derived from the proton translocating H+-V-ATPase subunit a were studied. An NMR structure was obtained in SDS micelles. Circular dichroism measurements indicated that the peptide formed a beta-sheet at high pH in octylglucoside, while it was 60% alpha-helical in SDS. These findings were explained using molecular dynamics simulations, which indicated that at high pH the peptide took a transmembrane position in SDS but was located in the interface region in octylglucoside. The combination of the NMR structure and the MD simulations allowed us to identify the residues that line the lumenal proton channel
24
Follit, John A. "Building the Cell's Antenna: Protein Targeting to the Ciliary Membrane: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/594.
Full textAbstract:
Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.
25
Jiang, Ying. "Transfer of the Ribosome-Nascent Chain Complex to the Translocon in Cotranslational Translocation: A Thesis." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/332.
Full textAbstract:
Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system.
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 sec61mutants impact different steps in the cotranslational translocation pathway.
An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.
26
Stone-Hulslander, Judith. "Mechanisms of Newcastle Disease Virus-Mediated Membrane Fusion: A Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/131.
Full textAbstract:
For many paramyxoviruses, including Newcastle disease virus (NDV), syncytia formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (23, 73). Because a potential interaction in paramyxovirus infected cells has never been clearly demonstrated, such an interaction was explored in Chapter 2 using coimmunoprecipitation and crosslinking. Both HN and F proteins could be precipitated with heterologous antisera after a five minute radioactive pulse as well as after a two hour chase in non-radioactive media, but at low levels. Chemical crosslinking increased detection of complexes containing HN and F proteins at the cell surface. After crosslinking, intermediate as well as high molecular weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after crosslinking resulted in the detection of complexes which electrophoresed in the stacker region of the gel, from 160-300 kD, at 150 kD and at 74 kD. Precipitates obtained with anti-F after crosslinking contained species which migrated in the stacker region of the gel, between 160-300 kD, at 120 kD and at 66 kD. The 3-4 discrete complexes ranging in size from 160-300 kD contained both HN and F proteins when precipitated with either HN or F antisera. That crosslinking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating crosslinking at early time points post infection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically crosslinked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the RER. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a subsequent change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion.
Chapter 3 explores the stalk region of the NDV HN protein, which has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (7, 51, 57). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled-coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha-helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic "a" position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.
27
Adhikari, Anirban. "Regulation of guanine nucelotide exchange in inhibitory G protein alpha subunit by activator of G protein signaling 3 and novel regulatory peptides." Embargoed access until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=114.
Full text
28
Weiß, Kerstin [Verfasser], Jörg [Akademischer Betreuer] Enderlein, and Sarah [Akademischer Betreuer] Köster. "Quantifying the diffusion of membrane proteins and peptides / Kerstin Weiß. Gutachter: Jörg Enderlein ; Sarah Köster. Betreuer: Jörg Enderlein." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2013. http://d-nb.info/1044739401/34.
Full text
29
Ye, Weihua. "One key to two doors : Dual targeting peptides and membrane mimetics." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-116817.
Full textAbstract:
A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an α-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a φχχφφ motif, where φ represents a hydrophobic/aromatic residue and χ represents any amino acid residue. In contrast, neither the amphiphilic helix nor φχχφφ motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction. In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form α-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: In press.
30
Mitsopoulos, Konstantinos. "The assembly of type III membrane proteins in Escherichia coli." Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310262.
Full text
31
Malmberg, Jennie. "The neuroanatomical expression profile of novel membrane proteins. : The effect of macronutrients on gene expression." Thesis, Mälardalen University, School of Sustainable Development of Society and Technology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-1105.
Full textAbstract:
Worldwide obesity is an increasing problem. Apart from the fact that obesity greatly impairs the health, quality and length of life for the affected individuals, it is also has the potential to become a major socioeconomic problem in a near future. However preventive actions require an understanding of the cause. Before the psychological influence on eating can be evaluated a profound understanding of the biological regulatory system and how this interacts with the food consumed is required. On the assumption that food consumption is regulated by interplay between food and genes, the food itself may influence the genes that regulate consumption, hence change the expression levels of the genes regulating food intake. To evaluate the interplay between food and gene expression, the project contained several parts, reflecting different aspects of the area of research. The feeding studies had in common that they were initial trials in a larger project. The results of these will be evaluated and used in combination with further studies. The mice typed for food preference illustrate the complexity of the feeding regulatory system by pointing out the differences between individuals even in a relatively small group of animals. Mice in general like food high in fat and here the animals that showed a preference for sugar also showed a significant increase in their intake of chow. Since chow consists mainly of carbohydrates the results might indicate a preference not for sucrose in particular but for carbohydrates in general. The effect this may have on other studies is still unclear as further studies are needed to determine whether the difference may be the result of an innate genetic difference. Leucine has been previously shown to reduce the total caloric intake. When given in combination with palatable food the addition of Leucine primarily reduced the intake of chow. From a dietary perspective this would translate to a preference to sweets and fast food at the expense of food with more nutritious content. The RT-PCR analysis’s gives clues to how the energy regulatory circuitry responds to the intake of selected macronutrients. When it comes to gene expression there is a significant effect of macronutrients on the gene expression levels. The common theme for many of the genes tested seems to be down regulation of satiety signals, as if to support over feeding on palatable diets and in many cases sucrose in particular. The intake of macronutrients such as sugar or fat has been showed to have an effect on the feeding regulatory circuitry, demonstrated by the change in gene expression levels. The response to said macronutrients is site specific which is clearly shown both by RTPCR analysis of samples from different parts of the brain, such as the brainstem or hypothalamus, and by immunohistochemistry of selected areas. The immunohistochemistry also confirms that the novel Oxytocin receptor-antagonist, who is injected IP, actually passes over the blood-brain barrier and has an actual affect on the regions of interest. The areas affected by the antagonist can be visualized and identified through the staining of active sites.
32
Gabriel, Luke R. "Dynamic Regulation at the Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels and Amphetamine-Sensitive Dopamine Transporters: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/725.
Full textAbstract:
Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.
33
Crowley, Jessica Lynn. "Role of Supervillin, a Membrane Raft Protein, in Cytoskeletal Organization and Invadopodia Function." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/406.
Full textAbstract:
Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia.
The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells.
Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.
34
Yunkun, Wu. "X-ray crystal structures of yeast heat shock proteins and mitochondrial outer membrane translocon member Tom70p." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/wu.pdf.
Full text
35
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.
Full textAbstract:
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.
36
Laliberte, Jason P. "Role of Host Cellular Membrane Raft Domains in the Assembly and Release of Newcastle Disease Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/360.
Full textAbstract:
Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions.
Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions.
Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity.
Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation.
The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.
37
Lai, Yinzhi. "CPLA2 key enzyme for astrocytic cell membrane phase property change induced by abeta /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/5992.
Full textAbstract:
Thesis (M.S.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 14, 2008) Includes bibliographical references.
38
Forbrig, Enrico [Verfasser], Peter [Akademischer Betreuer] Hildebrandt, Peter [Gutachter] Hildebrandt, Thomas [Gutachter] Gutsmann, and Peter [Gutachter] Hegemann. "Investigation of membrane-active peptides and proteins by vibrational spectroscopy / Enrico Forbrig ; Gutachter: Peter Hildebrandt, Thomas Gutsmann, Peter Hegemann ; Betreuer: Peter Hildebrandt." Berlin : Technische Universität Berlin, 2018. http://d-nb.info/1165138891/34.
Full text
39
Corey, Elizabeth Ann. "Characterization of the Relationship Between Measles Virus Fusion, Receptor Binding, and the Virus-Specific Interaction Between the Hemagglutinin and Fusion Glycoproteins: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/221.
Full textAbstract:
Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins.
Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins.
Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding.
Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established.
In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.
40
Gatlin, Jesse C. "Eicosanoid-mediated repellent signaling in the nerve growth cone : a role for the PKC substrate MARCKS /." Connect to full text at ProQuest Digital Dissertations. IP filtered, 2005.
Find full textAbstract:
Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.Typescript. Includes bibliographical references (leaves 123-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
41
Wang, Peng. "Studies on E. Coli Membrane Protein Biogenesis: Mechanism of Signal Peptide Peptidase A and the Influence of YiDC Depletion on Cellular Processes." Columbus, Ohio : Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view.cgi?acc%5Fnum=osu1243982038.
Full text
42
Malaby, Heidi L. H. "Identification of Molecular Determinants that Shift Co- and Post-Translational N-Glycosylation Kinetics in Type I Transmembrane Peptides: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/702.
Full textAbstract:
Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation.
To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini.
In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites.
Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates.
Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.
43
Malaby, Heidi L. H. "Identification of Molecular Determinants that Shift Co- and Post-Translational N-Glycosylation Kinetics in Type I Transmembrane Peptides: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/702.
Full textAbstract:
Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation.
To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini.
In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites.
Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates.
Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.
44
Harrington, Jane Colleen. "Regulation of Reactive Nitrogen Species (RNS) Metabolism and Resistance Mechanisms in Haemophilus influenzae: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/401.
Full textAbstract:
Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals.
FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.
45
Zhang, Jin. "Macromolecular Interactions in West Nile Virus RNA-TIAR Protein Complexes and of Membrane Associated Kv Channel Peptides." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/chemistry_diss/81.
Full textAbstract:
Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication
46
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.
Full textAbstract:
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.
47
Chen, Minyong. "Deciphering the Role of YidC in Bacterial Membrane Protein Insertion." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1039101039.
Full text
48
Cheng, Shiyuan. "Membrane assembly studies of subunit h : leader peptidase and m13 procoat proteins in Escherichia coli /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487777901659517.
Full text
49
Suwal, Shyam, and Shyam Suwal. "Fractionation of Peptides from Protein Hydrolysate by Electrodialysis with Filtration Membrane : process optimization, Fouling characterization and Control mechanisms." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26619.
Full textAbstract:
Des peptides bioactifs ont déjà été fractionnés par électrodialyse avec membrane de filtration (ÉDMF) à partir d’hydrolysats de sous-produits de crabe des neiges. L’optimisation des paramètres apparaît maintenant indispensable pour perfectionner le procédé. Ainsi, le taux de migration des peptides, leur sélectivité et l'évolution des paramètres électrodialytiques ont été étudiés pour différents paramètres (configuration, concentration en KCl et types de champ électrique). La configuration (2) de la cellule d’ÉDMF comprenant deux compartiments d'alimentation et un compartiment de récupération a démontré des valeurs de champ électrique local relativement stables par rapport à la configuration (1) constituée d’un compartiment d’alimentation et de deux compartiments de récupération. Des peptides contenant des glutamates, des aspartates, et des glycines ont été séparés avec la configuration 1 et des peptides composés d’arginines et de lysines avec la configuration 2. Un taux de migration peptidique de 13,76 ± 3,64 g/m2h a été obtenu par le maintien constant de la conductivité des solutions. La sélectivité a été accrue en augmentant la concentration en KCl de 1 à 5 g/L dans le compartiment de récupération. Une augmentation de la force ionique a amplifié la charge de surface, agrandissant ainsi la taille effective des pores et réduisant la couche d'hydratation de la membrane d’ultrafiltration. Toutefois, les membranes échangeuses d’anions et de cations ont été colmatées par des peptides et des acides aminés et détériorées pendant l’ÉDMF. Pour résoudre ces problèmes, l’effet de l’application du champ électrique pulsé (PEF) et de l'inversion de polarité (PR) a été étudié. Le taux de migration des peptides n'a pas été affecté sauf avec PR à 40 V. La sélectivité a été maximale avec PEF à 20 V. La dissociation de l'eau a été réduite tout en conservant les propriétés physico-chimiques des membranes grâce à l’application du PEF et de la PR par rapport au courant continu (DC). En outre, la plus faible quantité d'énergie a été consommée avec le PEF. Par conséquent, il a été possible d’optimiser la technologie d’ÉDMF du point de vue de l’efficacité énergétique, de la sélectivité peptidique et de l’encrassement membranaire grâce à l’application du PEF et tout en maintenant la conductivité électrique des solutions.Des peptides bioactifs ont déjà été fractionnés par électrodialyse avec membrane de filtration (ÉDMF) à partir d’hydrolysats de sous-produits de crabe des neiges. L’optimisation des paramètres apparaît maintenant indispensable pour perfectionner le procédé. Ainsi, le taux de migration des peptides, leur sélectivité et l'évolution des paramètres électrodialytiques ont été étudiés pour différents paramètres (configuration, concentration en KCl et types de champ électrique). La configuration (2) de la cellule d’ÉDMF comprenant deux compartiments d'alimentation et un compartiment de récupération a démontré des valeurs de champ électrique local relativement stables par rapport à la configuration (1) constituée d’un compartiment d’alimentation et de deux compartiments de récupération. Des peptides contenant des glutamates, des aspartates, et des glycines ont été séparés avec la configuration 1 et des peptides composés d’arginines et de lysines avec la configuration 2. Un taux de migration peptidique de 13,76 ± 3,64 g/m2h a été obtenu par le maintien constant de la conductivité des solutions. La sélectivité a été accrue en augmentant la concentration en KCl de 1 à 5 g/L dans le compartiment de récupération. Une augmentation de la force ionique a amplifié la charge de surface, agrandissant ainsi la taille effective des pores et réduisant la couche d'hydratation de la membrane d’ultrafiltration. Toutefois, les membranes échangeuses d’anions et de cations ont été colmatées par des peptides et des acides aminés et détériorées pendant l’ÉDMF. Pour résoudre ces problèmes, l’effet de l’application du champ électrique pulsé (PEF) et de l'inversion de polarité (PR) a été étudié. Le taux de migration des peptides n'a pas été affecté sauf avec PR à 40 V. La sélectivité a été maximale avec PEF à 20 V. La dissociation de l'eau a été réduite tout en conservant les propriétés physico-chimiques des membranes grâce à l’application du PEF et de la PR par rapport au courant continu (DC). En outre, la plus faible quantité d'énergie a été consommée avec le PEF. Par conséquent, il a été possible d’optimiser la technologie d’ÉDMF du point de vue de l’efficacité énergétique, de la sélectivité peptidique et de l’encrassement membranaire grâce à l’application du PEF et tout en maintenant la conductivité électrique des solutions.
Bioactive peptides were efficiently separated by using electrodialysis with filtration membrane (EDFM) from snow crab byproduct hydrolysate. Meanwhile, optimization of parameters is indispensable for scaling-up. The peptide migration rate and selectivity as well as evolution of electrodialytic parameters were studied with different parameters such as EDFM cell configuration, KCl concentration and type of electric field. The EDFM stack with two feed and one recovery compartments (configuration 2) has relatively stable electric field strengths (local) than the configuration with one feed and two recovery compartments (configuration 1). Peptides containing anionic amino acids: glutamic and aspartic acid as well as glycine and cationic amino acids: arginine and lysine were fractionated using configuration 1 and 2, respectively. Maintenance of solution conductivity upheld the local electric field and peptide migration throughout the treatment resulting in a higher peptide migration rate of 13.76±3.64 g/m2.h never observed so far. The selectivity of cationic peptides containing arginine and lysine increased significantly with increase in KCl concentration from 1 to 5 g/L. An increase in ionic strength amplified the surface charge density of filtration membrane subsequently increasing effective pore size and reducing hydration layer. However, both anion- and cation-exchange membranes were fouled by peptides and amino acids and were deteriorated during EDFM treatment. To address these problems, the effect of applying pulsed electric field (PEF) and polarity reversal (PR) was studied. The peptide migration rate was unaffected among PEF, PR and DC modes except with PR at 40 V. The selectivity of cationic peptides was maximum with PEF at 20 V. Fouling and water dissociation were significantly reduced and physicochemical properties of IEMs were better-protected with PEF and PR than DC. Moreover, the least amount of energy was consumed with PEF mode. Therefore, the parameters affecting EDFM process were optimized in terms of energy efficiency, selectivity and lower deterioration of membranes by applying PEF regime with configuration 2 and maintaining the constant electrical conductivity of solutions.
Bioactive peptides were efficiently separated by using electrodialysis with filtration membrane (EDFM) from snow crab byproduct hydrolysate. Meanwhile, optimization of parameters is indispensable for scaling-up. The peptide migration rate and selectivity as well as evolution of electrodialytic parameters were studied with different parameters such as EDFM cell configuration, KCl concentration and type of electric field. The EDFM stack with two feed and one recovery compartments (configuration 2) has relatively stable electric field strengths (local) than the configuration with one feed and two recovery compartments (configuration 1). Peptides containing anionic amino acids: glutamic and aspartic acid as well as glycine and cationic amino acids: arginine and lysine were fractionated using configuration 1 and 2, respectively. Maintenance of solution conductivity upheld the local electric field and peptide migration throughout the treatment resulting in a higher peptide migration rate of 13.76±3.64 g/m2.h never observed so far. The selectivity of cationic peptides containing arginine and lysine increased significantly with increase in KCl concentration from 1 to 5 g/L. An increase in ionic strength amplified the surface charge density of filtration membrane subsequently increasing effective pore size and reducing hydration layer. However, both anion- and cation-exchange membranes were fouled by peptides and amino acids and were deteriorated during EDFM treatment. To address these problems, the effect of applying pulsed electric field (PEF) and polarity reversal (PR) was studied. The peptide migration rate was unaffected among PEF, PR and DC modes except with PR at 40 V. The selectivity of cationic peptides was maximum with PEF at 20 V. Fouling and water dissociation were significantly reduced and physicochemical properties of IEMs were better-protected with PEF and PR than DC. Moreover, the least amount of energy was consumed with PEF mode. Therefore, the parameters affecting EDFM process were optimized in terms of energy efficiency, selectivity and lower deterioration of membranes by applying PEF regime with configuration 2 and maintaining the constant electrical conductivity of solutions.
50
Li, Xiaoman. "Study on memapsin 2 cleavage properties and its interacting proteins." Oklahoma City : [s.n.], 2010.
Find full text