Dissertations / Theses on the topic 'Beta barrel membrane proteins'
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1
Arjara, Gitrada Rees Douglas C. Gray Harry B. Richards John. "Refolding a beta-barrel membrane protein /." Diss., Pasadena, Calif. : California Institute of Technology, 2007. http://resolver.caltech.edu/CaltechETD:etd-05292007-061922.
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Pocanschi, Cosmin Lorin. "Folding and stability of beta-barrel membrane proteins from Gram-negative bacteria." [S.l. : s.n.], 2005. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-16869.
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Klein, Astrid. "A structural analysis of the TOB complex, the insertase for Beta-barrel proteins of the mitochondrial outer membrane." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-150786.
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Klein, Astrid [Verfasser], and Angelika [Akademischer Betreuer] Böttger. "A structural analysis of the TOB complex, the insertase for beta-barrel proteins of the mitochondrial outer membrane / Astrid Klein. Betreuer: Angelika Böttger." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1028490461/34.
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Miles, Jr George Emmett. "On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins." Texas A&M University, 2003. http://hdl.handle.net/1969.1/3952.
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Staphylococcal leukocidin pores are formed by the obligatory interaction
of two distinct polypeptides, one of class F and one of class S, making them
unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other
β-PFTs form homooligomeric pores. For example, the staphylococcal α-
hemolysin is a homoheptamer. Limited and controversial data exist on the
assembly and molecular architecture of the leukocidin pore.
In this work, biochemical and biophysical methods were used to
characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC)
components encoded by Staphylococcus aureus. I demonstrate that LukF and
LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In
addition, the pore-forming properties of recombinant leukocidin were
investigated with planar lipid bilayers. Although leukocidins and
staphylococcal α-hemolysin share partial sequence identity and related folds,
LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5
mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin
measured under the same conditions.
The subunit composition and stoichiometry of a leukocidin pore were
determined by two independent methods, gel shift electrophoresis and sitespecific
chemical modification during single channel recording. Four LukF and
four LukS subunits were shown to co-assemble into an octameric
transmembrane structure. The existence of an additional subunit in part
explains properties of the leukocidin pore, such as its high conductance.
Additionally, this is the first time that either technique has been applied
successfully to assess the composition of a heteromeric membrane protein. It is
also relevant to understanding the mechanism of assembly of β-PFT pores, and
suggests new possibilities for engineering these proteins.
In additional studies, the HlyII pore encoded by Bacillus cereus was found
to form a homoheptameric transmembrane pore with properties conforming in
general with those of other members of the class of β-PFTs. HlyII possesses
additional properties which make it an attractive candidate for applications in
biotechnology, such as an oligomer with a high thermal stability in the presence
of SDS and the ability of the pore to remain open at high transmembrane
potentials.
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Jorgenson, Matthew Allan. "A tale of two RLPAs : studies of cell division in Escherichia coli and Pseudomonas aeruginosa." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1342.
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Rare lipoprotein A (RlpA) has been studied previously only in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. In this thesis, we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in media of low osmotic strength. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from a ΔrlpA mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called "naked glycans"). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Collectively, these findings indicate RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall.
Our discovery that RlpA from P. aeruginosa is a lytic transglycosylase motivated us to reinvestigate RlpA from E. coli. We confirmed predictions that RlpA of E. coli is an outer membrane protein and determined its abundance to be about 600 molecules per cell. However, multiple efforts to demonstrate that E. coli RlpA is a lytic transglycosylase were unsuccessful and the function of this protein in E. coli remains obscure.
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Garrow, Andrew Gordon. "Search algorithms for transmembrane beta-barrel proteins." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427773.
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Pongprayoon, Prapasiri. "Molecular modelling of β-barrel outer membrane proteins." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:0ed0c22f-027e-4be1-a64c-0819888bbebc.
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In Gram-negative bacteria, the Outer membrane (OM) acts as a first barrier to screen unwanted compounds whilst enabling ions and very small solutes to diffuse into the cell. Most of nutrients and essential ions are effectively transported across a membrane via the outer membrane proteins (OMPs). The water-filled β- barrel OMPs are called porins. These pores are classified into two groups, non- specific and substrate-specific porins. Each of them has different mechanisms to facilitate its substrate translocation. To reveal the process of substrate permeation and selectivity in microscopic detail, molecular dynamics (MD) simulations and applications were performed in this thesis. The studies in this thesis focus on a series of classical porins. These proteins share similar feature where extracellular loop(s) (generally loop 3 (L3)) is folded into the middle of the pore and act as a constriction site which is important for substrate specificity and selectivity. The studies firstly concentrate on the structural properties and dynamics of the general trimeric porins, OmpC and OmpF whose sequences share 60% identity. OmpC and OmpF are found to have similar mechanism of latching loop (L2) to maintain trimeric stability. The smaller pore size allows OmpC to be more cation-selective than OmpF. Additionally, the major driving force for cation permeation in both porins is not from electrostatic properties. This differs from the phosphate-selective porin, trimeric OprP, where a phosphate diffusion depends on electrostatic interactions with positively charged pore-lining residues. The charge brush-like behavior of interior Arg and Lys residues plays a major role in phosphate selectivity. Also, the free energy profiles (PMF) reveal two key regions that are important for differentiating phosphate from other anions. The brush-like mechanism of OprP were also implanted to the simplified model pores in order to determine the possibility of transferring phosphate-selective properties of OprP to a model which may be useful for future design of nanopores. It is found that the duplication of functional residues and pore cavity can turn a model into the highly phosphate-selective pore. Importantly, the phosphate-binding affinity is dependent on the ability of the pore to interfere and occupy the hydration shell of a translocating phosphate where such ability can be maximized by an increase in sidechain flexibility. In case of uptake of more complex substrates, OpdK also employs a constriction site to select its substrate, aromatic vanillate (VNL) with total charge of -1. Unlike ion-specific porins, the free VNL is attracted by polar and aromatic interactions and sequentially directed through the periplasmic vestibule by charged residues insides the pore. The correct orientation of VNL on arrival is crucial for OpdK to recognize and enable the permeation process.
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Schiller, Stina. "Evolutive In-vitro-Adaption eines thermostabilen ([beta][alpha]8-barrel-Proteins [beta-alpha-8-barrel-Proteins] an die Katalyse einer abiotischen Reaktion." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=970732600.
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Tran, Thuong Van Du. "Modeling and predicting super-secondary structures of transmembrane beta-barrel proteins." Phd thesis, Ecole Polytechnique X, 2011. http://tel.archives-ouvertes.fr/tel-00647947.
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Les protéines transmembranaires canaux-β (TMBs) se trouvent dans les membranes externes des bactéries à Gram négatif, des mitochondries ainsi que des chloroplastes. Elles traversent entièrement la membrane cellulaire et exercent différentes fonctions importantes. Vu qu'il y a un petit nombre des structures des TMBs déterminées, en raison des difficultés avec les méthodes expérimentales, il est douteux que ces approches puis- sent bien trouver et prédire les TMBs qui ne sont pas homologues avec celles connues. Nous construisons un modèle de graphe pour la classification et la prédiction de structures super-secondaires permutées des TMBs à partir de leur séquence d'acides aminés, en se basant sur la minimisation d'énergie. Le modèle ne dépend essentiellement pas de l'apprentissage. Les algorithmes sont rapides, robustes avec des performances com- parables à celles des meilleures méthodes actuelles qui utilisent l'apprentissage. Cette méthode peut être donc utile pour le screening des génomes. Outre la performance de prédiction et de classification, cette étude donne une vue plus profonde de la structure des TMBs en tenant compte des contraintes physicochimiques des membranes biologiques. Les structures permutées prédites peuvent aussi aider à mieux comprendre le mécanisme du repliement des TMBs.
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Savojardo, Castrense <1981>. "Machine-learning methods for structure prediction of β-barrel membrane proteins." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5429/.
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Different types of proteins exist with diverse functions that are essential for living organisms. An important class of proteins is represented by transmembrane proteins which are specifically designed to be inserted into biological membranes and devised to perform very important functions in the cell such as cell communication and active transport across the membrane. Transmembrane β-barrels (TMBBs) are a sub-class of membrane proteins largely under-represented in structure databases because of the extreme difficulty in experimental structure determination. For this reason, computational tools that are able to predict the structure of TMBBs are needed. In this thesis, two computational problems related to TMBBs were addressed: the detection of TMBBs in large datasets of proteins and the prediction of the topology of TMBB proteins. Firstly, a method for TMBB detection was presented based on a novel neural network framework for variable-length sequence classification. The proposed approach was validated on a non-redundant dataset of proteins. Furthermore, we carried-out genome-wide detection using the entire Escherichia coli proteome. In both experiments, the method significantly outperformed other existing state-of-the-art approaches, reaching very high PPV (92%) and MCC (0.82). Secondly, a method was also introduced for TMBB topology prediction. The proposed approach is based on grammatical modelling and probabilistic discriminative models for sequence data labeling. The method was evaluated using a newly generated dataset of 38 TMBB proteins obtained from high-resolution data in the PDB. Results have shown that the model is able to correctly predict topologies of 25 out of 38 protein chains in the dataset. When tested on previously released datasets, the performances of the proposed approach were measured as comparable or superior to the current state-of-the-art of TMBB topology prediction.
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Hagan, Christine Lepicier. "Reconstitution of the E. Coli Membrane \(\beta\)-Barrel Assembly Machine from Purified Components." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10202.
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\(\beta\)-barrel membrane proteins perform important functions in the outer membranes of Gram-negative bacteria and in the mitochondria and chloroplasts of eukaryotes. Cellular machines that have been conserved from bacteria to humans assemble these proteins by an unknown mechanism. The components of the \(\beta\)-barrel assembly machine (Bam) in E. coli have been identified, but it has been difficult to study their function in vivo because they catalyze an essential process; mutations in proteins involved in the assembly pathway are often lethal or produce pleiotropic phenotypes that do not reveal the specific roles of the individual proteins. This study describes an in vitro reconstitution of the activity of the Bam complex and the use of this assay to determine how the Bam proteins contribute to the assembly of the complex itself. A sensitive assay for \(\beta\)-barrel assembly was developed using a substrate protein that has protease activity when it is folded. A peptide bond cleavage thereby reports on the conformational change the Bam complex catalyzes. This assay demonstrates that the Bam proteins dramatically increase the rate of \(\beta\)-barrel assembly without any external energy source. The structures of these proteins must inherently facilitate the folding and insertion process. The in vitro reconstitution was then adapted to study the assembly of the central component of the Bam complex, BamA. These studies reveal that the conserved domains of BamA catalyze the steps in the assembly process that are common in all organisms. The accessory components of the Bam complex adapt the mechanism of BamA to improve its efficiency and to allow it to handle a diverse set of substrates. The assembly of the Bam complex thus demonstrates how a cellular machine evolves to achieve generality and high efficiency. A structure of the Bam complex will be required to understand the molecular details of how substrate proteins are bound, folded into \(\beta\)-barrel structures, and inserted into the membrane. Initial efforts indicate that it will be possible to obtain such a structure. By combining structural and biochemical information garnered from the in vitro reconstitution, the general principles that guide the assembly of membrane \(\beta\)-barrels may be determined.Chemistry and Chemical Biology
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Shah, Aalok K. "Geometry Of Alpha And Beta Protein Structures." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/578419.
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Proteins have a wide array of essential functions: from serving as enzymatic catalysts to protecting the immune system as antibodies. Proteins spontaneously self-organize into specific, folded structures determined by their amino acid sequences and the interaction between molecular forces. Since the 3-dimensional structure into which they fold often relates to the specific function of the protein, much effort has been directed towards methods to predict the folded structure from a given sequence, with the hope of being able to understand protein functions from sequence information. The protein folding problem can be summarized as the attempt to understand the relationship between a protein sequence and a protein's geometric shape, or fold. Thus, there are two principal problems: given a sequence, what 3-dimensional form will the protein take (forward problem), and given a particular fold, what sequence or sequences code for that form (the inverse problem). In this work, models that represent folds as continuous structures are explored. Models of the two prevalent motifs in protein folds, α helices and β barrels, are developed using axially deformed tubes and surfaces of revolution. These models are then analyzed and used to develop coordinate models of known and unknown structures.
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Damaghi, Mehdi. "Characterizing the Functional and Folding Mechanism of β-barrel Transmembrane Proteins Using Atomic Force Microscope." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-114947.
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Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010).
Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent.
In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process.
We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG.
Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.
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Vincent, Karla Kristine. "Transactivation of Beta 2 Adrenergic Receptor by Bradykinin type 2 Receptor via heterodimerization." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37117.
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Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.
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Cohen, Alona. "Studies of regulated membrane trafficking /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1634379741&sid=7&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Bengtsson, Luiza. "Novel integral membrane proteins of the inner nuclear membrane characterization of LUMA native LAP 2[beta] [2beta] complexes /." [S.l. : s.n.], 2002. http://www.diss.fu-berlin.de/2002/100/index.html.
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Huang, Eugene Y. "The effect of enforced Notch signaling on TCR beta, positive, and negative selection of developing T cells /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8350.
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Miller, Jennifer Louise. "Engineering an ultra-thermostable β₁-adrenoceptor and its structure determination." Thesis, University of Cambridge, 2012. https://www.repository.cam.ac.uk/handle/1810/252260.
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Schmid, Katharina [Verfasser]. "Stability of the Outer Membrane Protein TtoA and Interaction with the Insertase TtOmp85 : FTIR studies on two β -barrel proteins / Katharina Schmid." Konstanz : Bibliothek der Universität Konstanz, 2016. http://d-nb.info/1126968994/34.
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Teo, Jia-Ling. "Presenilin-1 and TCF/[beta]-catenin signaling : effects on neuronal differentiation /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/9311.
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Häusler, Elisabeth [Verfasser], Franz [Akademischer Betreuer] Hagn, Michael [Gutachter] Sattler, and Franz [Gutachter] Hagn. "NMR structural and biochemical studies on the β-barrel membrane proteins OEP21 and hVDAC1 / Elisabeth Häusler ; Gutachter: Michael Sattler, Franz Hagn ; Betreuer: Franz Hagn." München : Universitätsbibliothek der TU München, 2020. http://d-nb.info/1238781551/34.
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Wiley, Jesse Carey. "Familial Alzheimer's disease mutations decrease gamma-secretase processing of beta amyloid precurson [sic] protein /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/4985.
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Laudon, Hanna. "Functional domains in the Alzheimer's disease-associated presenilin 1 protein /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-085-0/.
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Nilsberth, Camilla. "Distribution and pathophysiological role of amyloid precursor protein and presenilin 1 : characterization in rats and in vitro studies on the pathogenic arctic mutation /." Stockholm : Karolinska Univ. Press, 2002. http://diss.kib.ki.se/2002/91-7349-329-5/.
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陳嘉威 and Ka-wai Patrick Chan. "Transforming growth factor-{221}1 induces cell invasiveness via the downregulation of junctional adhesion molecule-A." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47151602.
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Hu, Jing. "Prediction of Protein Function and Functional Sites From Protein Sequences." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/292.
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High-throughput genomics projects have resulted in a rapid accumulation of protein sequences. Therefore, computational methods that can predict protein functions and functional sites efficiently and accurately are in high demand. In addition, prediction methods utilizing only sequence information are of particular interest because for most proteins, 3-dimensional structures are not available. However, there are several key challenges in developing methods for predicting protein function and functional sites. These challenges include the following: the construction of representative datasets to train and evaluate the method, the collection of features related to the protein functions, the selection of the most useful features, and the integration of selected features into suitable computational models. In this proposed study, we tackle these challenges by developing procedures for benchmark dataset construction and protein feature extraction, implementing efficient feature selection strategies, and developing effective machine learning algorithms for protein function and functional site predictions. We investigate these challenges in three bioinformatics tasks: the discovery of transmembrane beta-barrel (TMB) proteins in gram-negative bacterial proteomes, the identification of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs), and the identification of helix-turn-helix (HTH) motifs from protein sequence.
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Li, Xiaoman. "Study on memapsin 2 cleavage properties and its interacting proteins." Oklahoma City : [s.n.], 2010.
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Söderberg, Linda. "Characterization of the Alzheimer's disease-associated clac protein /." Stockholm : Karolinska institutet, 2005. http://diss.kib.ki.se/2005/91-7140-252-7.
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Almeida, Vitor Medeiros. "Identificação de domínios em β-glicosidases GH1 através da análise de sua estabilidade." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22082016-082333/.
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Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase.Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.
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Le, Marchand Tanguy. "Protein Dynamics by Solid-State NMR with Ultra-Fast Magic-Angle Spinning : from Microcrystals to Amyloid Fibrils and Membrane Proteins." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN023/document.
Full textAbstract:
La Résonance Magnétique Nucléaire (RMN) à l’état solide avec rotation à l’angle magique (MAS) est une technique de choix pour l’étude de la structure et de la dynamique de molécules biologiques peu ou non solubles. Un grand nombre d’approches ont été développées pour la reconstitution de structures tridimensionelles à partir de mesures précises de proximités internucléaires, ainsi que pour la détection de mouvements moléculaires avec une résolution atomique sur des échelles de temps couvrant plusieurs ordres de grandeur. Malgré d’impressionnants progrès, les études par RMN MAS sont cependant loin d’être réalisées en routine. Les déterminations structurelles et de dynamique sont souvent démontrées sur des préparations microcristallines modèles, mais sont encore rares pour des systèmes plus complexes tels que les fibrilles amyloïdes non cristallines ou les protéines trans-membranaires insérées dans des bi- couches lipidiques. Mon travail a pour objectif d’étendre les possibilités de la RMN MAS pour l’étude de systèmes biomoléculaires complexes dans différents états d’agrégation. Pour cela, j’ai exploité les possibilités uniques offertes par les hauts champs magnétiques (fréquence de Larmor du 1H 700, 800 et 1000 MHz) combinés avec les sondes MAS de dernières générations capables d’atteindre des vitesses de rotations supérieures à 60 kHz. Ces conditions expérimentales per- mettent d’augmenter la sensibilité de la RMN MAS à l’aide de la détection 1H à haute résolution et d’enrichir la palette de paramètres RMN rapporteurs de la dynamique des protéines. La première partie de cette thèse décrit le développement de nouvelles stratégies pour l’attribution des résonances du squelette de protéines, pour l’élucidation de structures, et pour l’étude de la dynamique du squelette peptidique et des chaînes latérales. Les méthodes présentées réduisent significative- ment les besoins en termes de temps expérimental, de quantités d’échantillon et de marquage isotopique, et permettent d’analyser par RMN des systèmes de plus hauts poids moléculaire. La seconde partie décrit l’application de la RMN MAS avec détection en 1H pour l’évaluation du rôle de la dynamique des protéines dans des processus tels que la formation de fibrilles amyloïdes et le fonctionnement de protéines membranaires. Une première application est l’étude de la tendance de la β-2 microglobuline humaine à former des fibrilles amyloïdes. Une comparaison de la dynamique du squelette peptidique de la protéine sauvage et du mutant D76N dans leur forme cristalline, ainsi que la détermination de propriétés structurales de la forme fibrillaire m’ont permis d’identifier la présence de repliements pathologiques et de formuler des hypothèses sur le mécanisme de formation des fibrilles. Finalement, la dynamique locale et globale de protéines membranaires dans des bicouches lipidiques a été étudiée. En particulier, le mécanisme d’action d’un transporteur d’alkanes, AlkL, de P. putida a été examiné dans un environnement lipidique. La détermination de paramètres pour la dynamique rapide (ps-ns) et lente (μs-ms) du squelette peptidique de la protéine en présence ou en absence de substrat met en évidence des acheminements possibles pour le transfert de molécules vers la membrane et jette les bases pour une meilleure compréhension du processusSolid-state NMR with magic angle spinning (MAS) has emerged as a powerful technique for investigating structure and dynamics of insoluble or poorly soluble biomolecules. A number of approaches has been designed for reconstructing molecular structures from the accurate measurement of internuclear proximities, and for probing motions at atomic resolution over timescales spanning several orders of magnitude. Despite this impressive progress, however, MAS NMR studies are still far from routine. Complete determinations, which are often demonstrated on model microcrystalline preparations, are still rare when it comes to more complex systems such as non-crystalline amyloid fibrils or transmembrane proteins in lipid bilayers. My work aimed at extending the possibilities of MAS NMR for applications on complex biomolecular systems in different aggregation states. For this, I exploited the unique possibilities provided by high magnetic fields (700, 800 and 1000 MHz 1H Larmor frequency) in combination with the newest MAS probes capable of spinning rates exceeding 60 kHz. These experimental conditions al- low to boost the sensitivity of MAS NMR through 1H detection at high resolution and to enrich the palette of probes for protein dynamics. The first part of the thesis reports on my contribution to the development of new strategies for backbone resonance assignment, for structure elucidation, and for investigation of backbone and side-chain dynamics. These methodologies significantly reduce the requirements in terms of experimental time, sample quantities and isotopic labeling, and enlarge the molecular size of systems amenable to NMR analysis. The second part describes the application of 1H detected MAS NMR to evaluate the role of protein dynamics in problems such as amyloid fibril formation and membrane protein function. I first addressed the amyloid fibril formation propensity of human beta-2 microglobulin, the light chain of the major histocompatibility complex I. I performed comparative studies of backbone dynamics of the wild type protein as well as a D76N mutant in crystals, and determined some of the structural features of the fibrillar form. This allowed to identify the presence of pathological folding intermediates and to formulate hypotheses on the mechanism of fibrils formation. Finally, I studied the local and global dynamics of membrane proteins in lipid bilayers. In particular, I investigated the mechanism of action of the alkane trans- porter AlkL from P. putida in lipid bilayers. The measurement of parameters for fast (ps-ns) and slow (μs-ms) backbone dynamics of the protein in presence or in absence of a substrate highlights possible routes for molecular uptake and lays the basis for a more detailed mechanistic understanding of the process
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Puig, Gomà-Camps Eduard. "Structural characterization of amyloid beta oligomers with functional links associated to Alzheimer's disease." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667258.
Full textAbstract:
Alzheimer’s disease (AD) is the most common form of dementia. It was first described in 1906 by Alois Alzheimer. Later on, in 1984 George Glenner and Colin Masters isolated the amyloid-beta (Aβ) peptide from a human brain and associated it to the disease. Since then the amyloid hypothesis has been a rather controversial matter discussed among the scientific community. This is because although Aβ has been targeted by the majority of the drugs in clinical trials not even one has been approved up to date: 13 have been discontinued and 10 are in phase 3 clinical trials. A possible explanation for these devastating numbers is the high complexity of the target due to the variety of aggregation forms that Aβ can adopt. Therefore, understanding the links between protein aggregation and neurotoxicity, and specially obtaining the 3D structures of the aggregates responsible for neurotoxicity is key to design effective diagnostic and therapeutic strategies. Unfortunately, this remains one of the most important unresolved issues in the field. The group of Dr. Carulla has been working on the hypothesis that Aβ interacts with the cell membrane leading to ionic dyshomeostasis. In order to study this scenario, the group has changed the paradigm and treated Aβ as a membrane protein and applied well known methodologies used to characterize this family of proteins to study Aβ. By doing so, the group has proved that Aβ is able to form a type of oligomer in the presence of detergent micelles which adopts a very specific and defined structure with characteristics of a β-barrel assembly and functions as a pore. They refer to these types of oligomer as β-Barrel Pore-Forming Oligomer (βPFO).
Hereby we present the work carried out to identify by using different biophysical techniques, the 3D structure of βPFO. As a starting point, we have used detergents to study the oligomerization process in a membrane mimetic environment. Micelles compared to other more native-like biomimetics environments based on lipids, will enable the application of novel mass spectrometry (MS) strategies and well-established solution NMR techniques thus providing high-resolution structural information. Since the accumulation of different amounts of Aβ in the membrane is a plausible scenario in the context of the disease, we have used different Aβ to detergent micelle ratios ([Aβ]:[M]) to study the role of this variable in the oligomerization process of Aβ. Throughout the work done we have optimized not only the ratio but also other conditions such as the buffer and the pH to modulate the preparation of samples enriched in defined oligomer populations.
To study the stoichiometry of βPFO, we used with Native Mass Spectrometry which proved to be an adequate technique to preserve the non-covalent interactions of our samples analyse them in the gas phase. One of the key parts of the project consisted in the screening of a wide range of non-ionic detergents compatible with MS. After this work as we were able to identify Pentaethylene Glycol Monooctyl Ether (C8E5) as the best candidate for our samples. To continue working with the different samples we implemented a new approach based on coupling size exclusion chromatography (SEC) directly to a SYNAPT G2. This approach has allowed us to establish that higher molecular weight oligomers are better preserved and therefore better detected as we increase the signal to noise ratio. This enabled us to study different points of the SEC chromatogram and therefore understand better the composition of our samples and our system. For the standard βPFO samples, we reported specific charge states for the octamer and tetramer species.
In parallel to complement the native-MS results, we have also worked to develop a method to analyse chemically cross-linked βPFOs by MALDI-MS. After a process of trials and optimizations we established a zero-length cross-linker (DMTMM) which allowed us to cross-link the βPFOs and detect again tetramer and octamer such as in the native-MS approach.
In order to assess the relevance and to potentially validate the standard βPFO preparation as a target for AD’s it is crucial to characterize the binding of the Nanobodies to the oligomer. This work will also give us the opportunity to generate Nanobodies that could recognize their specific structures in brain tissue and thus assess whether the oligomers proposed are related to AD’s and if so, evaluate them as new targets for AD. Moreover we are very interested in the potential use of these Nanobodies as novel diagnostics or therapeutics tools.La malaltia d'Alzheimer (AD) és la forma més comuna de demència. Va ser descrita per primera vegada el 1906 per Alois Alzheimer. Més endavant, al 1984, George Glenner i Colin Masters van aïllar el pèptid amiloide-beta (Aβ) d'un cervell humà i el van associar a la malaltia. Des de llavors, la hipòtesi amiloide ha estat un tema bastant controvertit discutit entre la comunitat científica. Una possible explicació és l’alta complexitat del sistema a causa de la varietat de formes d’agregació que Aβ pot adoptar. Per tant, entendre els vincles entre l'agregació de proteïnes i la neurotoxicitat, i especialment l'obtenció de les estructures 3D dels agregats responsables de la neurotoxicitat, és clau per dissenyar estratègies diagnòstiques i terapèutiques efectives. Malauradament, aquest tema continua sent un dels problemes pendents més importants. El grup de la Dra. Carulla ha estat treballant en la hipòtesi que l'Aβ interactua amb la membrana cel·lular que condueix a una deshomeostasi iònica. Per estudiar aquest escenari, el grup ha canviat el paradigma i ha tractat Aβ com a proteïna de membrana i aplicant tècniques biofísiques ben establertes per a caracteritzar proteïnes de membrana per tal d’estudiar Aβ. D'aquesta manera, el grup ha demostrat que Aβ és capaç de formar un tipus d'oligòmers en presència de micel·les de detergent que adopten una estructura molt específica i definida amb capacitat de formar porus a través de membranes lipídiques. Es refereixen a aquest tipus d’oligòmers com a oligòmers formadors de porus barril β (βPFO). A la present tesi doctoral, presentem l’estudi realitzat per identificar mitjançant diferents tècniques biofísiques, l'estructura 3D de βPFO. Hem utilitzat detergents per estudiar el procés d’oligomerització en un entorn mimètic de membrana. Les micel·les en comparació amb altres entorns biomimètics basats en lípids, permeten l'aplicació d’estratègies d'espectrometria de masses (MS) i de ressonància magnètica nuclear (RMN) ben establertes, proporcionant així informació estructural d'alta resolució. Atès que l’acumulació de diferents quantitats d’Aβ a la membrana és un escenari plausible en el context de la malaltia, hem utilitzat diferents relacions de micel·les Aβ a detergents ([Aβ]: [M]) per estudiar el paper d’aquesta variable en l’oligomerització. procés d'Aβ.
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Chang, Tao-Hsin. "Structural studies of Norrin dependent Wnt/beta-catenin signaling." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:a9f2d687-e8ee-4f26-9a8a-12efb87445ae.
Full textAbstract:
Norrin is a secreted cystine-knot growth factor that plays critical roles in vascular development in the brain, retina, and cochlea, as well as the uterus. Although Norrin is unrelated to the lipid-modified morphogens Wnts, Norrin activates the canonical Wnt/β-catenin pathway by binding to receptor Frizzled4 cysteine-rich domain (Fz4-CRD) and co-receptors of low density lipoprotein receptor related protein 5/6 ectodomain (Lrp5/6-ECD) in conjunction with Tetraspanin-12 (Tspan-12). Like Wnts, Norrin has limited extracellular diffusion properties as a result of associating with heparan sulfate proteoglycans (HSPGs). Mutations lead to inherited disordered retinal vascularization diseases such as Norrie disease, familial exudative vitreoretinopathy and coats' disease. However, the molecular mechanism of how Norrin initiates signalling by engagement with Fz4, Lrp5/6, and HSPGs has remained unresolved. Here, novel strategies for protein production of recombinant human Norrin and Fz4-CRD as well as the complex are developed. The crystal structures of Norrin and its complex with Fz4-CRD, plus complex bound with the heparin mimic sucrose octasulphate, and unliganded structures of Fz4-CRD are presented. These structural data together with biophysical and cellular assays not only reveal the Fz4 and Lrp5/6 binding sites on distinct patches of the Norrin surface, but also indicate the HSPGs binding site on Norrin and Fz4-CRD as well as providing a framework to explain numerous disease-related mutations. Structural comparison with Xenopus Wnt8 in complex with mouse Fz8-CRD provides molecular insights for our understanding of ligand-receptor binding specificity and promiscuity, which has important implications for developing therapeutic strategies against Norrin dependent retinal disorders, and cancers caused by abnormal Wnt signaling.
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O'Sullivan-Murphy, Bryan M. "Contribution of WFS1 to Pancreatic Beta Cell Survival and Adaptive Alterations in WFS1 Deficiency: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/590.
Full textAbstract:
Diabetes mellitus comprises a cohort of genetic and metabolic diseases which are characterized by the hallmark symptom of hyperglycemia. Diabetic subtypes are based on their pathogenetic origins: the most prevalent subtypes are the autoimmune-mediated type 1 diabetes mellitus (T1DM) and the metabolic disease of type 2 diabetes mellitus (T2DM). Genetic factors are major contributory aspects to diabetes development, particularly in T2DM where there is close to 80% concordance rates between monozygotic twins. However, the functional state of the pancreatic β cell is of paramount importance to the development of diabetes. Perturbations that lead to β cell dysfunction impair insulin production and secretion and precede diabetes onset.
The endoplasmic reticulum (ER) is a subcellular organelle network of tubes and cisternae with multifaceted roles in cellular metabolism. Alterations to ER function such as those begotten by the accumulation of misfolded and unfolded ER client proteins upset the ER homeostatic balance, leading to a condition termed ER stress. Subsequent sensing of ER stress by three ER transmembrane proteins, initiates an adaptive reaction to alleviate ER stress: this is known as the unfolded protein response (UPR). Divergent cascades of the UPR attempt to mitigate ER stress and restore ER homeostasis: Failing that, the UPR initiates pro-apoptotic pathways. The demand of insulin production on the β cell necessitates the presence of a highly functional ER. However, the consequence of dependence on the ER for insulin synthesis and secretion portends disaster for the functional state of the β cell. Disturbances to the ER that elicit ER stress and UPR activation causes β cell dysfunction and may lead to apoptosis. There are numerous well-characterized models of ER stress-mediated diabetes, including genetic mutations in UPR transducers and insulin. Recently, polymorphisms in Wolfram syndrome 1 (WFS1), an ER transmembrane protein involved in the UPR, were suggested to contribute to T2DM risk.
In this thesis, one of the highlighted WFS1 polymorphism, H611R, was examined to identify its contribution to β cell function and viability, and hence, diabetes risk. It was revealed that augmentation of WFS1 expression increased insulin secretion and cellular content. In addition, WFS1 protected β cells against ER stress-mediated dysfunction, with a more pronounced effect in the WFS1-R611 protective allele. Subsequent gene expression analysis identified netrin-1 as a WFS1-induced survival factor.
As a contributory factor to diabetes progression, ER stress and UPR are potential drug and biomarker targets. In this dissertation, a novel UPR-regulating microRNA (miRNA) family was uncovered in ER stressed, WFS1-deficient islets. These miRNAs, the miR-29 family, are induced in WFS1 -/- islets as a possible adaptive alteration to chronic ER stress conditions, and indirectly decreases the expression of UPR transducers, while directly targeting downstream ER stress-related pro-apoptotic factors. Collectively, this work extends the function of WFS1 as a protective factor in the pancreatic β cell through the induction of netrin-1 signaling. Additionally, it further strengthens the role of miRNA as regulatory members of the UPR which contribute to cell survival.
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Lipson, Kathryn L. "The Role of Endoplasmic Reticulum Stress Signaling in Pancreatic Beta Cells: a Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/363.
Full textAbstract:
Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin.
Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress.
In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis.
In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.
This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity.
This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.
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Aguilar, Mónica Alejandra Pavez. "Caracterização molecular da resistência aos carbapenêmicos em enterobactérias isoladas em hospitais brasileiros." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-28092009-144325/.
Full textAbstract:
Introdução: Após o surgimento e disseminação das β-lactamases (BL) de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenem, ertapenem) têm sido considerados a terapia de escolha pela estabilidade apresentada contra estas enzimas. Infelizmente, em 2005, o primeiro caso de infecção fatal por um isolado de Klebsiella pneumoniae resistente aos carbapenêmicos foi relatado em nosso país. A partir deste, novos casos de infecção, inclusive por outros gêneros da família Enterobacteriaceae como Enterobacter, Providencia e Escherichia, começaram a surgir. Como mecanismo de resistência aos carbapenêmicos, a expressão de enzimas carbapenemases tem sido mundialmente relatada, enquanto que, a impermeabilidade associada à produção de enzimas do tipo AmpC ou ESBL tem sido esporádica. Com relação à mobilização dos determinantes genéticos de resistência, elementos móveis como integrons e plasmídios têm sido associados. O presente trabalho teve como objetivo caracterizar os mecanismos de resistência aos carbapenêmicos, sua mobilização genética e disseminação clonal em amostras clínicas de enterobactérias isoladas em diversos hospitais brasileiros. Material e métodos: Foram estudadas 28 cepas recuperadas de oito centros hospitalares descritas como resistentes ao imipenem. A caracterização fenotípica foi realizada por: i) determinação da CIM na presença e ausência de inibidores de BL, ii) bioensaio para produção de BL e iii) SDS-PAGE para investigar a ausência de porinas. A confirmação genotípica da resistência mediada por β-lactamases foi realizada por PCR e seqüenciamento e a sua localização plasmidial foi estudada por transformação. Por último, a tipagem molecular foi realizada pela técnica de ERIC-PCR, sendo confirmada pela técnica de PFGE. Resultados: 25 cepas apresentaram resistência para carbapenêmicos (imipenem MIC 8-128 µg/mL), todas com perfil de multiresistência incluindo cefoxitina (CIM90 ≥32 µg/mL). Foram identificados três determinantes de resistência, entre eles, a produção de carbapenemases de tipo MBL (IMP-1) e a enzima KPC-2, recentemente descrita, sendo emergente no país. O mecanismo mais prevalente nas amostras estudadas foi a impermeabilidade de membrana associada à expressão de enzimas do tipo AmpC (CMY-2 plasmidial para E. coli e AmpC cromossômica no caso de Enterobacter aerogenes), as quais mostraram uma contribuição significativa para a resistência aos carbapenêmicos. Dos 28 isolados, 18 apresentaram a perda da porina de 36 kDa, responsável pela entrada de antimicrobianos na bactéria, como os carbapenêmicos. Tanto os genes blaKPC-2 e blaCMY-2 foram transferidos com êxito para E. coli DH10B, confirmando sua localização plasmidial. A co-produção de carbapenemase ou enzimas do tipo AmpC com ESBL do tipo CTX-M foi confirmada em 68% dos isolados. A tipagem molecular mostrou uma disseminação clonal para os isolados carregando determinantes IMP-1 e as enzimas do tipo AmpC cromossômica e plasmidial. Ao contrário, isolados expressando KPC não foram clonalmente relacionadas. Conclusão: A caracterização de resistência apresentada neste trabalho demonstrou uma mudança no perfil de resistência da família Enterobactériaceae devido à sua versatilidade para a aquisição de novos mecanismos de resistência, como sua adaptação aos ambientes hostis. A perda da porina foi o mecanismo mais freqüente nesta família e a co-produção de BL foi um evento associado. Finalmente, os dados obtidos na tipagem molecular denotaram uma disseminação majoritariamente clonal na cidade de São Paulo, com exceção das cepas produtoras de KPC-2, cuja presença tem sido relatada em outras cidades do país, sugerindo a participação de uma transferência horizontal.Introduction: After emergence, and dissemination of extended spectrum β-lactamases (ESBL) in members of the Enterobacteriaceae family, carbapenem antibiotics (imipenem, meropenem, ertapenem) have been the therapy of choice, since they are stable to ESBL hydrolysis. Unfortunately, in 2005, the first fatal case of infection by carbapenem-resistant Klebsiella pneumoniae was related in our country. From this episode, new infection cases, including by other genders of Enterobacteriaceae such as Enterobacter, Providencia and Escherichia, began to appear. Regarding carbapenem resistance mechanisms, expression of carbapenem hydrolyzing enzymes has been worldwide reported, whereas interplay between impermeability and AmpC or ESBL production has been sporadic. Furthermore, integrons and plasmids have been associated with mobilization of genetic determinants. The aim of this study was to characterize the mechanisms of resistance to carbapenems, their genetic mobilization and clonal dissemination in enterobacterial isolates recovered from clinical samples in Brazilian hospitals. Material and methods: 28 imipenem-resistant isolates recovered from 8 hospital centres were studied. Phenotypic profiles were characterized by: i) MIC of carbapenems in the presence/absence of β-lactamase inhibitors; ii) bioassay for β-lactamase production; iii) SDS-PAGE to investigate absence of outer membrane porins (OMPs). Molecular characterization of β-lactamase-mediated resistance was made by PCR and DNA sequencing and their plasmid localization was evaluated by transformation. Finally, epidemiological typing was performed by ERIC-PCR, being confirmed by PFGE. Results: 25 isolates were confirmed as being resistant to imipenem (MIC 8-128 µg/mL), exhibiting a multidrug-resistant profile, including to cefoxitin (MIC90 ≥32 µg/mL). Two main mechanism of resistance were identified: i) hydrolysis of carbapenem by class B (IMP-1-like MBL) and class A (KPC-2) enzymes, (the latter being recently reported in our country), and ii) outer membrane impermeability associated to AmpC enzyme production (plasmid-mediated CMY-2 for E. coli and chromosomal AmpC for E. aerogenes), which was the most prevalent mechanism found. Eighteen of 28 isolates lacked 36kDa OMP, which is responsible for uptake of carbapenem antibiotics. The blaKPC-2 and blaCMY-2 genes were successful transferred to E. coli DH10B, confirming the plasmid location of both genes. Co-production of carbapenemases or AmpC and CTXM enzymes was confirmed in 68% of isolates, and molecular typing showed clonal dissemination of IMP-1-, plasmid AmpC- and chromosomal AmpC-producing isolates. Otherwise, KPC-2-producing isolates were not clonally related. Conclusion: The characterization of resistance mechanisms to carbapenems, in this study, reveals a change in the resistance patterns among Enterobacteriaceae family members in Brazilian hospitals, due to versatility of isolates to acquire new resistance determinants, which it has favoured the adaptation to hostile environments. Lack of 36 kDa OMP was the most frequent resistance mechanism, being associated to co-production of β-lactamases. Finally, molecular typing denote a clonal dissemination of imipenem-resistant isolates in Sao Paulo city, with exception of KPC-2-producing isolates, which have been described in other Brazilian cities, suggesting a horizontal gene transfer.
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Fan, Yan, Ping Ping Chen, Ying Li, Kui Cui, Daniel M. Noel, Elizabeth D. Cummins, Daniel J. Peterson, Russell W. Brown, and Meng-Yang Zhu. "Corticosterone Administration up-Regulated Expression of Norepinephrine Transporter and Dopamine Β-Hydroxylase in Rat Locus Coeruleus and Its Terminal Regions." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/955.
Full textAbstract:
Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.
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Arjara, Gitrada. "Refolding a beta-barrel membrane protein." Thesis, 2007. https://thesis.library.caltech.edu/2245/1/01_Title.pdf.
Full textAbstract:
The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a ?-barrel membrane protein. The amphiphilic, ?-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus.
While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate ?-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate.
Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.
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Pocanschi, Cosmin L. [Verfasser]. "Folding and stability of β-barrel [beta-barrel] membrane proteins from Gram-negative bacteria / vorgelegt von Cosmin L. Pocanschi." 2006. http://d-nb.info/98292268X/34.
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Schiller, Stina [Verfasser]. "Evolutive In-vitro-Adaption eines thermostabilen (βα)8-barrel-Proteins [beta-alpha-8-barrel-Proteins] an die Katalyse einer abiotischen Reaktion / vorgelegt von Stina Schiller." 2004. http://d-nb.info/970732600/34.
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Damaghi, Mehdi. "Characterizing the Functional and Folding Mechanism of β-barrel Transmembrane Proteins Using Atomic Force Microscope." Doctoral thesis, 2012. https://tud.qucosa.de/id/qucosa%3A26923.
Full textAbstract:
Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010).
Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent.
In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process.
We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG.
Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.:Table of Contents
INTRODUCTION:1
1.1 THE FIRST UNIT OF LIFE STARTED WITH MEMBRANE:1
1.2.1 CELL MEMBRANE STRUCTURE: 2
1.3 MEMBRANE PROTEINS:3
1.3.1 α-‐HELICAL MEMBRANE PROTEINS:5
1.3.2 β-‐BARREL MEMBRANE PROTEIN:5
1.4 MEMBRANE PROTEINS FOLDING:12
1.4.1 MODELS FOR α-‐HELICAL MEMBRANE PROTEIN FOLDING:13
1.4.2 MODELS FOR β-‐BARREL MEMBRANE PROTEIN FOLDING:15
1.5. GATING STUDY OF MEMBRANE PROTEINS:18
ATOMIC FORCE MICROSCOPY:19
2.1 ATOMIC FORCE MICROSCOPE:19
2.1.1 HISTORY:19
2.1.2 PRINCIPLE:19
2.1.3 THE CANTILEVER:20
2.1.4 AFM MODES 23
2.2 SINGLE-‐MOLECULE FORCE SPECTROSCOPY:25
2.2.1 DYNAMIC FORCE SPECTROSCOPY,(DYNAMIC SMFS):27
2.3 WHAT IS THE ADVANTAGE OF USING ATOMIC FORCE MICROSCOPY IN
MEMBRANE PROTEIN STUDIES?:29
FOLDING MECHANISM OF OMPG:31
3.1 UNFOLDING PATTERN: ONEβ-‐HAIRPIN AFTER THE OTHER:31
3.1.1 OUTER MEMBRANE PROTEIN G (OMPG):31
3.1.2 MECHANICAL UNFOLDING PATHWAYS OF THE MEMBRANE β-‐BARREL PROTEIN OMPG:33
3.1.3 MATERIAL AND METHODS:34
3.1.4 RESULTS AND DISCUSSION:41
3.2 REFOLDING PATTERN: ONE Β-‐HAIRPIN AFTER THE OTHER:48
3.2.1. EXPLORING REFOLDING PATHWAYS AND KINETICS OF THE MEMBRANE Β-‐BARREL PROTEIN OMPG:48
3.2.2 EXPERIMENTAL PROCEDURES:49
3.2.3 RESULTS:50
3.2.4 DISCUSSION:52
INTERACTION STUDIES:59
4.1 PH-‐DEPENDENT INTERACTIONS GUIDE THE FOLDING AND GATE THE TRANSMEMBRANE PORE OF THE β-‐BARREL TRANSMEMBRANE PROTEIN OMPG:59
4.1.2 INTRODUCTION:59
4.1.2 EXPERIMENTAL PROCEDURES:61
4.1.3 RESULTS AND DISCUSSION:62
4.2 DUAL ENERGY LANDSCAPE: THE FUNCTIONAL STATE OF THE OUTER MEMBRANE β-‐BARREL PROTEIN OMPG MOLDS ITS UNFOLDING ENERGY LANDSCAPE:67
4.2.1 INTRODUCTION:67
4.2.2 EXPERIMENTAL PROCEDURES:71
4.2.3 RESULTS AND DISCUSSION:74
4.2.3.1 FUNCTIONAL STATE OF OMPG DIRECTS ITS UNFOLDING ROUTE:74
4.2.3.2 QUANTIFYING THE UNFOLDING ENERGY BARRIERS OF OMPG IN THE CLOSED AND OPEN CONFORMATIONS:75
4.2.3.3 TRANSITION STATE DISTANCES OF UNFOLDING ENERGY BARRIERS:77
4.2.3.4 ACTIVATION FREE ENERGY OF Β-‐STRANDS AND Β-‐HAIRPINS:79
4.2.3.5 MECHANICAL PROPERTIES OF OMPG:83
4.2.3.6 MAPPING THE UNFOLDING ENERGY LANDSCAPES OF OMPG IN THE OPEN AND CLOSED STATES:85
4.2.4 CONCLUSION:86
OUTLOOK:89
5.1 INTRODUCTION:89
5.2 INTERACTION STUDY AND
UNFOLDING ENERGY LANDSCAPE:90
5.3 MEMBRANE PROTEINF OLDING:92
REFRENCES:96
ABBREVIATIONS:110
SYMBOLS:111
PUBLICATIONS:113
ACKNOWLEDGMENT:114
DECLARATION: 115
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Hayat, Sikander [Verfasser]. "Sequence based methods for the prediction and analysis of the structural topology of transmembrane beta barrel proteins / von Sikander Hayat." 2010. http://d-nb.info/1009599496/34.
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Nelson, Christopher David. "Characterization of Beta-arrestin-Modulated Lipid Kinase Activities for Diacylglycerol and Phosphatidylinositol 4-Phosphate." Diss., 2007. http://hdl.handle.net/10161/206.
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KIM, JUN TAE. "Static and dynamic adsorption of [beta]-lactoglobulin on polymeric membrane surface and development of novel membranes by surface modification." 2007. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.17106.
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Wang, Leo D. "Investigation into the roles of the cytoplasmic tails of Ig-alpha and Ig-beta in B cell development /." 2003. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3077081.
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Williamson, Ritchie, A. Usardi, D. P. Hanger, and B. H. Anderton. "Membrane-bound beta-amyloid oligomers are recruited into lipid rafts by a fyn-dependent mechanism." 2008. http://hdl.handle.net/10454/6237.
Full textAbstract:
Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.
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"Identification and characterization of novel FE65-interacting proteins." 2009. http://library.cuhk.edu.hk/record=b5896580.
Full textAbstract:
Cheng, Wai Hang.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 76-88).
Abstract also in Chinese.
Acknowledgement --- p.i
摘要 --- p.iii
List of Abbreviations --- p.iv
List of Figures --- p.vi
List of Tables --- p.vii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- FE65 --- p.1
Chapter 1.1.1 --- FE65 Protein Family and Their Structures --- p.2
Chapter 1.1.1.2 --- PTB domains --- p.5
Chapter 1.1.2 --- Expression Pattern of FE65 Proteins --- p.6
Chapter 1.1.3 --- FE65 Family-Transgenic Animals --- p.7
Chapter 1.1.4 --- Interacting Partners of FE65 --- p.8
Chapter 1.1.4.1 --- "APP, APLPl and APLP2" --- p.9
Chapter 1.1.4.2 --- LRP1 and ApoEr2 --- p.10
Chapter 1.1.4.3 --- c-Abl --- p.11
Chapter 1.1.4.4 --- Mena and EVL --- p.11
Chapter 1.1.4.5 --- Tip60 --- p.12
Chapter 1.1.4.6 --- SET --- p.12
Chapter 1.1.4.7 --- Estrogen Receptor a --- p.13
Chapter 1.1.4.8 --- Teashirt --- p.13
Chapter 1.1.4.9 --- CP2/LSF/LBP1 --- p.13
Chapter 1.1.4.10 --- Dexra sl --- p.14
Chapter 1.1.4.11 --- P2X2-receptor subunit --- p.14
Chapter 1.1.4.12 --- Tau --- p.15
Chapter 1.1.4.13 --- Notchl --- p.15
Chapter 1.1.4.14 --- Alcadein --- p.16
Chapter 1.1.4.15 --- CD95/Fas/Apo -1 ligand --- p.16
Chapter 1.1.4.16 --- p68 subunit of pre -mRNA cleavage and polyadenylation factor Im (p68 CFIm) --- p.17
Chapter 1.1.4.17 --- Ataxinl --- p.17
Chapter 1.1.5.1 --- FE65 as an adaptor protein --- p.20
Chapter 1.1.5.2 --- FE65 and Alzheimer´ةs disease --- p.20
Chapter 1.1.5.3 --- Transcriptional / Post-transcriptional regulation --- p.22
Chapter 1.1.5.4 --- Apoptosis and cell cycle regulation --- p.23
Chapter 1.1.5.5 --- Neuronal positioning and cell migration --- p.23
Chapter 1.1.5.6 --- Learning and memory --- p.25
Chapter 1.2 --- Objectives --- p.26
Chapter Chapter 2 --- Investigation of the interaction between FE65 and Arf6 --- p.27
Chapter 2.1 --- Materials --- p.27
Chapter 2.1.1 --- DNA contructs --- p.27
Chapter 2.1.2 --- Cell culture --- p.27
Chapter 2.1.3 --- Immunoblotting --- p.28
Chapter 2.1.4 --- Miscellaneous --- p.28
Chapter 2.2 --- Methods --- p.29
Chapter 2.2.1 --- Preparation of Escherichia coli competent cells --- p.29
Chapter 2.2.2 --- DNA preparation with Intron Plasmid DNA --- p.30
Chapter 2.2.3 --- DNA preparation with Macherey-Nagel NucleoBond Xtra Midi --- p.30
Chapter 2.2.4 --- DNA preparation by the alkaline lysis method --- p.31
Chapter 2.2.5 --- Spectrophotometric analysis of DNA --- p.32
Chapter 2.2.6 --- Agarose gel electrophoresis --- p.32
Chapter 2.2.7 --- Cell culture and transfection --- p.33
Chapter 2.2.8 --- Bacterial GST-pull down assay --- p.33
Chapter 2.2.9 --- GST-pull down assay for testing direct interaction between FE65 and Arf6 --- p.34
Chapter 2.2.10 --- Mammalian GST-pull down assay --- p.35
Chapter 2.2.11 --- Immunoprecipitation --- p.36
Chapter 2.2.12 --- SDS-PAGE --- p.36
Chapter 2.2.13 --- Immunoblotting --- p.39
Chapter 2.3 --- Results --- p.40
Chapter 2.3.1 --- Interaction between Arf6 and FE65 --- p.40
Chapter 2.3.2 --- Determination of the interacting domain of FE65 with Arf6 --- p.43
Chapter 2.3.3 --- Determination if FE65 and Arf6 interact directly --- p.45
Chapter Chapter 3 --- Production of Antisera against Arf6 and Immunostaining of FE65-Arf6 --- p.47
Chapter 3.1 --- Materials --- p.47
Chapter 3.1.1 --- Protein expression and purification --- p.47
Chapter 3.1.2 --- Immunization and harvest of antisera --- p.48
Chapter 3.1.3 --- Immunostaining --- p.48
Chapter 3.2 --- Methods --- p.48
Chapter 3.2.1 --- Protein expression and purification --- p.48
Chapter 3.2.2 --- Bradford assay --- p.50
Chapter 3.2.3 --- Immunization --- p.50
Chapter 3.2.4 --- Antibody purification --- p.51
Chapter 3.2.5 --- Immunostaining --- p.52
Chapter 3.3 --- Results --- p.53
Chapter 3.3.1 --- Recombinant Arf6 expression and purification --- p.53
Chapter 3.3.2 --- Titering of antisera --- p.57
Chapter 3.3.3 --- Determination of antisera specificity --- p.59
Chapter Chapter 4 --- Discussion --- p.68
Chapter Chapter 5 --- Future Perspectives --- p.73
References --- p.76
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Gunasekaran, K. "Stereochemical Analysis On Protein Structures - Lessons For Design, Engineering And Prediction." Thesis, 1997. http://etd.iisc.ernet.in/handle/2005/2140.
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