Kliknij ten link, aby zobaczyć inne rodzaje publikacji na ten temat: Outer membrane protein A (OmpA).
Rozprawy doktorskie na temat „Outer membrane protein A (OmpA)”
Utwórz poprawne odniesienie w stylach APA, MLA, Chicago, Harvard i wielu innych
Sprawdź 50 najlepszych rozpraw doktorskich naukowych na temat „Outer membrane protein A (OmpA)”.
Przycisk „Dodaj do bibliografii” jest dostępny obok każdej pracy w bibliografii. Użyj go – a my automatycznie utworzymy odniesienie bibliograficzne do wybranej pracy w stylu cytowania, którego potrzebujesz: APA, MLA, Harvard, Chicago, Vancouver itp.
Możesz również pobrać pełny tekst publikacji naukowej w formacie „.pdf” i przeczytać adnotację do pracy online, jeśli odpowiednie parametry są dostępne w metadanych.
Przeglądaj rozprawy doktorskie z różnych dziedzin i twórz odpowiednie bibliografie.
1
Kaye, Elena Cortizas. "The Function of Outer Membrane Protein A (OmpA) in Yersinia pestis". Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_theses/58.
Pełny tekst źródłaStreszczenie:
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.
2
Schesser, Bartra Sara Celinda. "Outer membrane proteins of Yersinia pestis : Ail and OmpA". Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-33956.
Pełny tekst źródłaStreszczenie:
A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.
3
Al-Akash, Ahmed M. "Increased expression of ompA, ompX, dedA, and gutS genes in Enterobacter sp. YSU in the presence of selenite". Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607517925584702.
Pełny tekst źródła
4
McCallan, Lyanne Mary. "Differentiation of Campylobacter jejuni on the basis of outer membrane protein (OMP) patterns". Thesis, University of Ulster, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422191.
Pełny tekst źródła
5
Gerlach, Lisa [Verfasser]. "BamB facilitates folding of outer membrane protein A (OmpA) via interactions of its β-propeller with the membrane surface and via a conformation change induced by phosphatidylglycerol / Lisa Gerlach". Kassel : Universitätsbibliothek Kassel, 2020. http://d-nb.info/1204016488/34.
Pełny tekst źródła
6
Kevin, Gross. "Characterization of a fourU RNA thermometer in the ompA gene of Shigella dysenteriae". Ohio University Honors Tutorial College / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1367582905.
Pełny tekst źródła
7
Dahlstrand, Rudin Arvid, i John Burstedt. "The importance of OuterMembrane Protein A in SerumResistance in Aggregatibacteractinomycetemcomitans serotype astrain D7SS". Thesis, Umeå universitet, Institutionen för odontologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-143904.
Pełny tekst źródłaStreszczenie:
The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is primarily associatedwith aggressive forms of periodontal disease. Additionally, it has occasionally been found to causemetastatic infections in non-oral sites. This requires the ability to evade the bactericidal activity ofthe complement system of the humoral immune system. Outer membrane proteins, namely,Omp100 and OmpA have been connected to normal human serum resistance for several bacteriaspecies. The objective of this study was to investigate if serum-resistant ompA mutants can beobtained, and to detect changes in OMP expression. We used A. actinomycetemcomitansserotype a strain D7SS and D7SS ompA knockouts. The strains were incubated in 50 % NHS.This resulted in a substantial decrease of survival among D7SS ompA knockouts. D7SS ompAknockouts were exposed to 50 % NHS once more to confirm stable serum resistance. 13 out of14 tested clones showed growth, indicating that serum resistant ompA mutants could begenerated. SDS-PAGE gel of extracted outer membrane vesicles revealed an additional proteinband of approximately 34 kDa in at least 4 of 5 tested serum resistant ompA mutants. This proteinband has been analyzed in the laboratory, and according to LC-MS/MS it contains an OmpAhomologue, which has been named OmpA2. We conclude that OmpA2 expression might be amajor mechanism for serum survival in A. actinomycetemcomitans serotype a strain D7SS ompAknockouts.
8
Easton, Donna Meredith, i n/a. "Functional and Antigenic Characterisation of the Moraxella catarrhalis protein M35". University of Canberra. n/a, 2008. http://erl.canberra.edu.au./public/adt-AUC20081217.083105.
Pełny tekst źródłaStreszczenie:
This thesis reports the characterisation of a novel outer membrane protein (OMP)
from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is
structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and
OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16
antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse
clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides)
with only one isolate (ID78LN266) having base variations that resulted in amino acid
substitutions.
A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266
significantly affected antibody recognition, indicating that loop 3 contains an immunodominant
B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266
was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin
channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be
a potential mechanism for evasion of host immune responses targeted to M35, potentially
explaining the high degree of conservation across isolates.
A series of recombinant proteins were constructed to analyse the binding to M35 of
antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3-
specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and
that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro
and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35
lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective
than the full consensus M35 by both these measures. It therefore appears that while the
majority of antibodies raised against M35 are specific for loop 3 these antibodies do not
mediate anti-M. catarrhalis actions.
Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane
protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons
between these mutant strains and their wildtype parent strains initially led to the
hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis,
however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid
seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion
mutant strains, presumably to compensate for the lack of M35. M35 was also found to
be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating
that it is an essential functional protein for colonisation of the mucosal surface.
9
Li, Huanyu. "Structural studies of the BAM complex, OmpU outer membrane protein and lipoprotein N-acyl transferase in Gram-negative bacteria". Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/68718/.
Pełny tekst źródłaStreszczenie:
Structural studies of membrane proteins represent a significant challenge in the field owing to their hydrophobic nature, unstable property and resistance to be crystallized. In Gramnegative bacteria, membrane proteins contribute to the characteristic membranous architecture composed of an asymmetric layer of outer membrane (OM) and a symmetric inner cytoplasmic membrane (IM). Outer membrane proteins (OMPs) play essential roles in nutrient uptake, protein transport, outer membrane assembly, and pathogenesis of Gram-negative bacteria. In Escherichia coli, nearly all the outer membrane proteins are inserted into the outer membrane by the β-barrel assembly machinery (BAM), which contains one conserved membrane protein BamA and four lipoproteins BamBCDE. The individual protein structures of the BAM complex have been reported, but the mechanism of OMP assembly by the BAM complex is halted by a lack of structure of the whole complex. During the course of the collaborative BAM complex project, I participated in structural studies of the BAM complex and generated high resolution crystallographic diffraction data that contributes to one of the two determined structures of the BAM complex, and the structural insights have enlightened understanding of the in vivo insertion mechanism. Of diverse types of β-barrel OMPs that are inserted into the OM by the BAM complex, an outer membrane protein called OmpU from Vibrio cholerae is a potential virulence factor in addition to its porin identity with undefined atomic structure. I determined the crystal structure of this OMP, in which the long and flexible extracellular loop L4 and a novel Nterminal coil in the pore lumen provide direct structural evidence underlying its particular functions. The symmetric lipid bilayer of IM accommodates an even more diverse array of IMPs composed of the contrasting dominance of α-helices. Three IMPs are responsible for conducting post-translational modifications of lipoproteins in Gram-negative bacteria, a class of proteins destined to reside on the periplasmic side of either the IM or the OM via acyl chains post-translationally linked to the N-terminal cysteine residues, and they are called phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt), Prolipoprotein signal peptidase (Lsp) and apolipoprotein N-acyltransferase (Lnt). Structural studies were carried out on Lnt but unsuccessful in determining the atomic structure. Recent structures of Lnt reported during the course of this project are consistent with earlier biochemical studies that piloted the understanding of its function and further elucidate the molecular mechanisms of its primary acyl-transfer function.
10
Holmqvist, Erik. "Macromolecular Matchmaking : Mechanisms and Biology of Bacterial Small RNAs". Doctoral thesis, Uppsala universitet, Mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171642.
Pełny tekst źródłaStreszczenie:
Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.
11
Thotakura, Gangadaar. "Structural prediction analysis of ehrlichia chaffeensis outer membrane proteins, p28 Omp-14 and p28 Omp-19 assessed by circular dichrosim and porin assays". Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/9098.
Pełny tekst źródłaStreszczenie:
Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Roman Reddy R. Ganta
Ehrlichia chaffeensis, a Gram-negative organism belonging to the order Rickettsiales, is responsible for an emerging infectious disease in humans, the human monocytic ehrlichiosis. E. chaffeensis also infects several other vertebrate hosts including dogs, goats, coyotes and white tailed deers. This organism is transmitted by an infected tick, Amblyomma americanum. The exact pathogenic mechanisms involved for the persistence of the pathogen in vertebrate hosts are still unclear. E. chaffeensis protein expression varies significantly in vertebrate and tick hosts. Differentially expressed proteins include the immunodominant outer membrane proteins encoded by the p28-Omp multigene locus. The p28-Omp 14 is expressed primarily in tick cells and the p28-Omp 19 is the major expressed protein in macrophages both under in vitro and in vivo conditions. The objective of this study is to prepare recombinant proteins and use them to assess the secondary structures and protein functions. The protein sequences were analyzed with the aid of bioinformatics programs to make structural predictions. The analysis suggested the presence of eight β barrel structures for both the p28-Omp proteins. The coding sequence of the p28-Omp genes were cloned and over expressions of proteins in in E. coli was accomplished by using the plasmid expression construct, pET28. The proteins were purified to near homogeneity and used to refold using detergents to mimic native protein structure in the bacterial outer membrane. Refolding of proteins was analyzed by two methods; SDS-PAGE and Circular Dichroism. The Circular dichroism spectroscopy analysis suggested the formation of β-sheet structures of proteins in micelles formed with the detergents. β-sheet structures may have been formed with the hydrophobic domains of the protein imbedded in the micelles. The hydrophilic segments (predicted by bio informatics analysis) may be exposed to the aqueous phase. The recombinant proteins were also iii used to prepare proteoliposomes and tested for the porin activity. The analysis demonstrated the porin activity for both p28-Omp 14 and 19 recombinant proteins by using mono-, di- and tetra- saccharides as well as for amino acid L-glutamine. This study forms the basis for initiating studies to compare the structural difference between the two differentially expressed proteins of E. chaffeensis.
12
Barlow, Ann Katherine. "Neisseria meningitidis : the class 1 outer membrane protein". Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280415.
Pełny tekst źródła
13
McBride, Heidi May. "Protein import into and across the mitochondrial outer membrane". Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40395.
Pełny tekst źródłaStreszczenie:
Protein import into the mitochondria is a result of a series of sequential binding interactions between a mitochondrial targeting signal and the translocation machinery of both mitochondrial membranes. The targeting signals contained within protein of the outer membrane are distinct from those which target proteins to other subcompartments. The transmembrane domain of the yeast outer membrane receptor protein yTom70 is capable of both targeting and inserting the protein into the outer membrane. The efficiency of this process is increased by the addition of a positively-charged region preceding the transmembrane region. These two structural domains co-operate to form a signal-anchor sequence selective for the outer membrane, since this is the first membrane encountered by the targeting signal.Consistent with this model, the signal-anchor sequence of the outer membrane protein yTom70 is also capable of importing into the inner membrane of mitochondria when the outer membrane is selectively removed. Import into the inner membrane requires the presence of an electrochemical potential across the lipid bilayer. Import proceeds in the absence of $ Delta Psi$ only when constructs are used which lack the positively-charged amino terminal region of the signal-anchor sequence. These results suggest that the positively-charged presequence leads the transmembrane domain into the import machinery and that $ Delta Psi$ is required to clear this region in order that the distal transmembrane region can enter the translocation pathway.
The charged N-terminal 10 residues of yTom70 are incapable of directing import into intact mammalian mitochondria, however, are able to efficiently direct import into the matrix of yeast mitochondria or mammalian mitoplasts. This potentially cryptic signal is excluded from intact mammalian mitochondria due to the presence of the receptor protein Tom20, since replacement of yeast Tom20 with mammalian Tom20 confers the mammalian phenotype onto yeast. These results suggest that receptor proteins may also have the ability to screen potentially cryptic signals from distal components of the outer and inner membrane translocation machinery.
14
Menon, Sailesh. "Characterization of a Fusobacterium necrophorum subspecies necrophorum outer membrane protein". Kansas State University, 2014. http://hdl.handle.net/2097/18128.
Pełny tekst źródłaStreszczenie:
Master of ScienceDepartment of Biomedical Sciences
Sanjeev K. Narayanan
Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.
15
Shand, Geoffrey H. "Antibiotic resistance and outer membrane protein antigens of Pseudomonas aeruginosa". Thesis, Aston University, 1985. http://publications.aston.ac.uk/12475/.
Pełny tekst źródła
16
Sanchez, Katheryn Marie. "Ultraviolet resonance Raman and fluorescence studies of folded and unfolded conformations of the membrane protein OmpA". Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/ucsd/fullcit?p3397317.
Pełny tekst źródłaStreszczenie:
Thesis (Ph. D.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed April 7, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
17
See, Sarah Bihui. "Outer membrane protein immunity to Pasteurella pneumotropica and the interaction of allergy". University of Western Australia. School of Paediatrics and Child Health, 2010. http://theses.library.uwa.edu.au/adt-WU2010.0103.
Pełny tekst źródłaStreszczenie:
[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cellmediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.
18
Ferris, Shirley. "Antibody responses to the major outer membrane protein of Chlamydia trachomatis". Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295880.
Pełny tekst źródła
19
Schiffrin, Robert. "Roles of periplasmic chaperones and BamA in outer membrane protein folding". Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15952/.
Pełny tekst źródłaStreszczenie:
A defining feature of living things is that they have an inside and an outside, and in order for all living cells to survive, whether they are part of a blue whale or a unicellular microscopic organism, they must have mechanisms to mediate exchange with their environment. Food and energy enters the cell, and material must also leave, such as the waste products of metabolism, or virulence factors from pathogenic organisms. Lipid membranes define these boundaries, but it is membrane proteins that mediate the exchange. Although lipid bilayers can self-assemble in vitro, the assembly of complex biological membranes containing proteins requires energy and careful coordination. The work presented in this thesis examines the biogenesis pathway of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. OMPs are synthesised on cytosolic ribosomes, translocated across the inner membrane, then chaperoned across the periplasm, before folding and insertion into the OM. While OMPs can fold spontaneously into lipid membranes, this process is too slow to be biologically relevant, so a dedicated folding catalyst, the β-barrel assembly machinery (BAM) complex, is required at the OM. Recent genetic, structural and biochemical investigations have increased our understanding of OMP assembly, but key questions remain, including: How do periplasmic chaperones bind and release OMP substrates? What are the roles and interactions of BAM subunits? What is the molecular mechanism by which BAM folds and inserts OMPs? Here, an assay was developed to monitor OMP folding kinetics in vitro using intrinsic fluorescence in low concentrations of urea (0.24 M). This allowed comparison of the real-time folding behaviour of different OMPs under the same conditions for the first time (Chapter 3). The assay was then successfully extended to include OMP assembly factors, including the periplasmic chaperones Skp and SurA, and BamA, the principal component of the BAM complex, to obtain the following key results: Investigations into the interactions between Skp and OMPs of varying size (tOmpA, PagP, OmpT, OmpF and tBamA) revealed that greater Skp:OMP ratios are required to prevent the folding of 16-stranded OMPs compared with smaller 8-stranded OMPs. Supported by ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modelling and molecular dynamics simulations, the results imply a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp to protect a single substrate from aggregation (Chapter 4). Addition of further folding factors to the assay demonstrated that the model OMP tOmpA can be released and folded from its complex with Skp by BamA, possibly recapitulating an in vivo assembly pathway. BamA consists of a β-barrel membrane-embedded domain and soluble periplasmic domains, and while the release activity was shown to located in the membrane domain, the activity was greatest when full-length BamA was present. By contrast, SurA was not able to release tOmpA from Skp under the conditions employed, arguing against a sequential chaperone model (Chapter 5). Next, kinetic studies were used to investigate the mechanism of OMP folding catalysis by the BAM complex. The effect of hydrophobic mismatch between the BamA β-barrel and the membrane was examined by monitoring the folding of tOmpA into liposomes containing lipids of different chain lengths in the presence or absence of BamA. The results showed that BamA has a greater catalytic effect in lipids with longer chain lengths, with the largest rate enhancement achieved in bilayers with a hydrophobic thickness close to that of the OM. The results establish the importance of hydrophobic mismatch in the mechanism by which OMPs are folded in vivo, which may be influenced by local thinning of the membrane and increases in lipid disorder in the vicinity of the BAM complex (Chapter 5). Finally, based on the results obtained in this project, and consideration of the currently available literature, a new 'barrel elongation' model is proposed for the mechanism of OMP assembly by the BAM complex (Chapter 6). The OMP assembly pathway is an attractive target for novel antibacterials given that it is surface located, highly conserved, and essential in clinically important pathogens. Understanding the molecular mechanisms of OMP biogenesis factors will facilitate the development of drugs targeting this pathway. The work described in this thesis provides new insights into the mechanisms of OMP assembly, using a wide range of biochemical and biophysical techniques, thereby contributing to the development of this fast-moving and fascinating field.
20
Huysmans, Gerard Herman Marleen. "On the folding mechanism of the bacterial outer membrane protein PagP". Thesis, University of Leeds, 2008. http://etheses.whiterose.ac.uk/6752/.
Pełny tekst źródłaStreszczenie:
Membrane proteins represent an important class of macromolecules that play critical roles in many biological processes. The energetic principles underlying their stability, however, are not well understood. To address this deficiency, the kinetics and thermodynamics of the folding of the Escherichia coli outer membrane protein PagP were investigated by exploiting the ability of this polypeptide to refold into detergent micelles and into artificial lipid membranes. Investigations using the latter enabled the contributions to the folding process of both the protein sequence and of the bilayer lipid oomposition to be discerned.
21
Webb, Dianne, i n/a. "Nontypeable Haemophilus influenzae outer membrane protein analysis, isolation, characterisation and vaccine potential". University of Canberra. Applied Science, 1998. http://erl.canberra.edu.au./public/adt-AUC20061110.105953.
Pełny tekst źródłaStreszczenie:
Heterogeneity in immunodominant outer membrane proteins has been
proposed as a significant factor in the failure of an NTHi infection to induce
immune protection against subsequent infections. This study has examined
the vaccine potential of three outer membrane proteins in an attempt to
identify conserved regions that could be targeted by an immune response
after vaccination. The three proteins investigated were: TbpB, P5 and P48
(HI0164). The optimal route of immunisation in clearing a bolus inoculum
of NTHi to the lung in the rat has been shown to be a combination of gut
sensitisation with a respiratory boost and this regime was used in the present
study.
A panel of NTHi isolates was assessed to determine the frequency with
which strains were able to bind transferrin and thus be targeted by a TbpBspecific
immune response. A high proportion of strains was able to bind
transferrin with similar frequencies in isolates associated with infection and
those from normal throat swabs. A protocol was developed to purify
nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase
(GST)-rTbpB fusion protein and Glutathione-Sepharose affinity
chromatography. Mucosal-directed immunisation with rTbpB significantly
enhanced clearance of an NTHi challenge to the lung, however, whilst
rTbpB-specific antibodies were cross-reactive on Western immunoblots, the
cross-reactivity was variable in both transferrin binding inhibition assays and
bactericidal activity. This suggested that the rTbpB-specific humoral response
would be variable in the recognition of heterologous NTHi isolates.
The secondary structure of P5 has been controversial with several reports
suggesting that P5 was a fimbrin protein composed of coiled coils. In this
present study the interstrain variation in P5 amongst isolates from diverse
anatomical sites, as well as computer prediction methods and
spectrophotometric analysis, generated a model of P5 based on the
homologous E. coli protein, OmpA. This model suggested a B-barrel
conformation with no evidence of coiled coils. Synthetic peptides
corresponding to conserved regions of P5 that were thought to be surface
exposed, as well as a region (H3) with some homology to a protective epitope
in the P. aeruginosa protein, OprF, were then combined with a
"promiscuous" T cell epitope from the measles virus F protein (MVF) and
used for immunisation studies. Whilst variable protection was seen with the
peptides, the MVF/H3 peptide was the most efficacious of the antigens
assessed in this study in enhancing clearance of NTHi. This occurred in the
absence of detectable peptide- or PS-specific antibody leading to the
suggestion that cell mediated responses may have played an important role
in enhancing clearance in this model. The highly conserved nature of the
region in P5 represented by the H3 peptide suggests that further study should
be focused on this peptide as a potential NTHi vaccine candidate.
The last antigen, P48, is homologous to a A. pleuropneumoniae antigen,
AopA, which has been proposed to have potential as a vaccine component
against pleuropneumonia in pigs. Sequence analysis of the gene encoding
P48 from several isolates showed that this protein was well conserved.
Recombinant P48 was purified from a GST-rP48 fusion protein and used for
immunisation, which also conferred significant protection. However,
immunisation with rP48 was not as efficacious as immunisation with the
MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody
titres, no bactericidal activity could be detected indicating that bactericidal
antibody had not contributed to the observed clearance. In addition, the rP48-
specific serum IgG was predominantly of the IgG2a isotype suggesting that
Thl cell mediated responses had been induced by immunisation with rP48.
22
Arbing, Mark A. "Electrophysiological properties of porin, the major outer membrane protein of Haemophilus influenzae". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38146.
Pełny tekst źródłaStreszczenie:
Haemophilus influenzae (Hi) is a Gram-negative bacteria that is the causative agent of bacterial meningitis. The outer membrane (OM) of Gram-negative bacteria functions as a selectively permeable barrier. The exchange of small hydrophilic solutes between the external environment and the periplasm is mediated by large water-filled channels, porins. Charged residues of the pore determine the functional properties of the protein, which include: ion conductance, ionic selectivity, and voltage gating.To study the properties of the porin (341 amino acids; Mr 37,782) of Haemophilus influenzae type b (Hib), purified porin was subjected to chemical modification. The covalent modification of lysine residues with succinic anhydride (SA; Mr 100.08) results in charge reversal. The addition of up to 12 succinate groups per porin molecule was identified using electrospray ionization mass spectrometry (MS). Tryptic digestion of the modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight MS identified the sites of succinylation. The majority of modified lysines were positioned in surface-located loops, numbers 1 and 4 to 7. When the electrophysiological properties of SA-modified porin were analyzed in planar lipid bilayers (PLBs) and compared to Hib porin it was found that the single channel conductance was increased, while the threshold for voltage gating was decreased. The addition of extra negative charges increase the single channel conductance of Hib porin and function as voltage sensors.
Selected lysine residues that were found to be modified with SA were substituted with glutamic acid using site-directed mutagenesis. Single point mutations were made in a residue assigned to the barrel lumen and to three residues in each of loops 4 and 6. The mutant Hib porins had increased single channel conductances relative to wild-type Hib porin. Voltage gating of mutant Hib porins was altered by the introduction of negative charges into loops 4 and 6 and in the barrel lumen. Previous experiments had implicated surface-exposed loop 4 in voltage gating. This study ascribes a role for residues in loop 6 and a residue within the barrel lumen in the changes that accompany pore closure.
Hi strains causing infection in cystic fibrosis patients are capable of persistent infection despite prolonged antibiotic treatment with beta-lactam antibiotics. During the course of infection porin properties may be altered due to the changes in porin sequences that are attributed to antigenic drift. The electrophysiological properties of four porins from CF patient-derived Hi strains were characterized to examine changes in porin properties arising from persistent infection of the CF lung. The clinical Hi porins displayed altered channel properties that included increased voltage sensitivity and single channel conductances that were either greater or smaller than that of Hib porin. The decreased single channel conductance of one of the porins was associated with an increase in the minimal inhibitory concentration of the antibiotics novobiocin and streptomycin. These results demonstrate a porin-mediated decrease in OM permeability as an antibiotic resistance mechanism for Hi.
23
Carter, Michael William. "The major outer membrane protein gene of Chlamydia as a phylogenetic chronometer". Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336071.
Pełny tekst źródła
24
Eneroth, Elina, i Astrid Karlsson. "A role of Aggregatibacter actinomycetemcomitans Outer Membrane Protein 100 in Serum Resistance?" Thesis, Umeå universitet, Institutionen för odontologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128759.
Pełny tekst źródłaStreszczenie:
The disease periodontitis is an inflammatory response to the oral bacterial microflora. The Gram-negative bacterium Aggregatibacter actinomycetemcomitans has been specifically associated with the aggressive type of periodontitis. This species has a number of virulence factors that can contribute to host cell death, tissue inflammation, bone resorption, and colonization advantage relative to other microbes. A. actinomycetemcomitans has defense mechanisms against killing by the complement system (a part of our immune system). In the alternative pathway of complement activation, a protein called Factor H is the main regulator by having the ability to inhibit the complement reactions in three separate ways. Binding of Factor H to the bacterial surface was earlier shown to promote survival in human serum of an A. actinomycetemcomitans serotype d strain. This binding was caused by the outer membrane protein Omp100. The aim of this study was to establish whether Omp100 has a similar role in other serotypes of A. actinomycetemcomitans. For this we examined the serotype a strains D7S and D7SS, and their omp100 knockout mutants, which were generated in this laboratory. Western immunoblotting was used to compare a possible amount difference of the protein in each serotype. By incubating the bacterial strains in human serum, our results showed that lack of Omp100 did not have an obvious negative effect on the serum survival rate in strain D7S nor D7SS. We therefore concluded that omp100 was not required for serum resistance in these serotype a strains, and suggest that there might be another protein or factor in this serotype that is more important for serum resistance.
25
Morgan, Cecilia A. "Treponema pallidum repeat protein K and heterologous protection against syphilis /". Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/9300.
Pełny tekst źródła
26
Oliveira-Fry, Anna Maria, i s9911120@student rmit edu au. "Identification of Legionella outer membrane proteins for the development of a biosensor". RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080513.140744.
Pełny tekst źródłaStreszczenie:
Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.
27
Dahan, David. "Biophysics of porin, the major outer membrane protein of Haemophilus influenzae type b". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0025/NQ29916.pdf.
Pełny tekst źródła
28
Srikumar, Ramakrishnan. "Topology of porin, the major outer membrane protein of Haemophilus influenzae type b". Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40261.
Pełny tekst źródłaStreszczenie:
Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis in infants. A protein called porin of 341 amino acids, M$ rm sb{r}$ 37,782 daltons, is located in the outer membrane of Hib and allows for the diffusion into the periplasmic space of small solutes up to a molecular mass of 1400 daltons.Based on parameters of hydrophilicity and amphiphilicity a model for Hib porin was generated. The model proposed an organization of sixteen anti-parallel $ beta$-strands that traverse the outer membrane, eight long loops that connect the $ beta$-strands on one side and short turns on the other side.
By flow cytometry, six out of a panel of nine monoclonal antibodies against Hib porin recognized amino acid sequences at the cell surface. Hib porin was purified and subjected to chemical and enzymatic digestions. The fragments were immunoblotted; N-terminal sequencing identified boundaries of fragments. C-terminal deletions of Hib porin generated in the baculovirus expression system identified C-terminal boundaries of monoclonal antibody reactivities.
To map precisely the primary sequences to which these monoclonal antibodies bound, overlapping hexapeptides for the entire sequence of Hib porin were synthesized. These studies identified two surface-exposed regions in the mature sequence of Hib porin, amino acid residues 162-172 and 318-325. In the Hib porin model, these regions correspond to loops 4 and 8, respectively. Two regions between residues 112-126 (loop 3) and residues 148-153 were buried or inaccessible at the surface of the outer membrane.
Recombinant Hib porin was expressed in Bacillus subtilis. The biophysical and immunological properties of this lipooligosaccharide-free recombinant Hib porin were compared with those of native Hib porin.
In order to examine the role of loop 3, site-directed mutagenesis of the cloned Hib porin gene was undertaken. Six or twelve amino acid deletions in loop 3, expressed in a porin deletion strain, showed significant increase in sensitivities to several anti-microbial agents as compared to wild-type Hib porin. Deletion of twelve amino acids showed more pronounced phenotypes than deletion of six amino acids. Such mutagenesis experiments provided support to the notion that loop 3 in Hib porin folds back into the pore and produces a constriction of the channel.
29
Dahan, David. "Biophysics of porin : the major outer membrane protein of Haemophilus influenzae type b". Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42009.
Pełny tekst źródłaStreszczenie:
Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis in infants. Located in the outer membrane, Hib porin (342 amino acids, M$ rm sb{r}$ 37,782 daltons) forms channels that allow for the diffusion into the periplasm of small solutes up to a molecular mass of 1400 daltons.Based on predictions of porin secondary structure, a model of Hib porin was generated. The model proposed sixteen membrane spanning $ beta$-strands connected by eight long loops on one side and eight short $ beta$-turns on the other side. To refine further this model, the epitopes of nine monoclonal antibodies specific for Hib porin were mapped precisely. Surface-exposure of these epitopes was determined by flow cytometry of intact Hib cells. Our studies identified two surface-exposed regions of Hib porin: amino acids 162 to 172, and 318 to 325. These two regions correspond to loops 4 and 8 in the Hib porin model.
Three naturally occurring Hib porin variants were purified, reconstituted into planar bilayers, and analyzed for their voltage dependency. Substitutions at Arg166 residue, which is localized to loop 4, were associated with a lowered threshold potential for the voltage gating of Hib porin. This surface-exposed loop was therefore implicated in the conformational changes that are postulated to occur during voltage gating.
Lipooligosaccharide (LOS) binds tightly to Hib porin. To generate large amounts of Hib porin devoid of LOS, the ompP2 gene was cloned into an expression vector of Bacillus subtilis. Recombinant Hib porin was produced in large quantities and it aggregated into inclusion bodies. Under denaturing conditions, recombinant porin was purified to homogeneity and subsequently refolded. To assess the fidelity of refolding of recombinant porin, four criteria were used: spectroscopic properties, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by monoclonal antibody Hb-2. We conclude that in the absence of LOS, recombinant porin was refolded into a functional form, its structure closely resembling the native state.
30
Ward, Stephen John. "Characterisation and immunogenicity of recombinant class 1 outer membrane protein of Neisseria meningitidis". Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296140.
Pełny tekst źródła
31
Huang, Shouxiong. "Genetic and Antigenic Characterization of the Major Outer Membrane Protein of Campylobacter Jejuni". The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1046991783.
Pełny tekst źródła
32
Wyllie, Susan. "Structural and functional characterisation of the major outer membrane protein of Chlamydia psittaci". Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/22762.
Pełny tekst źródłaStreszczenie:
The major outer membrane protein (MOMP) of Chlamydia shares several biochemical properties with classical porin proteins. To directly test the "porin channel" hypothesis at the molecular level, MOMP was reconstituted into planar lipid bilayers where it gave rise to "triple-barrelled" channels which were modified by an anti-MOMP neutralising monoclonal antibody. These observations are consistent with the well characterised homo-oligomeric nature of MOMP previously revealed by biochemical analysis, and the "triple-barrelled" behaviour of other porins. MOMP channels were weakly anion selective (PCl/PK ~ 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates, and explain why some anti-MOMP antibodies neutralise infection. In order to undertake more detailed studies of the MOMP structure/function relationship, recombinant MOMP from both C. psittaci and C. pneumoniae have been cloned and expressed. The recombinant proteins were functionally reconstituted in planar lipid and analysed at the single channel level. Both form porin-like ion channels that are functionally similar to the native protein. The C. psittaci recombinant porin was modified by the same anti-MOMP neutralising monoclonal antibody that effected the native protein. In contrast to the native protein, both recombinant C. psittaci (PCl/K ~ 0.38) and C. pneumonia (PCl/PK ~ 0.49) proteins were marginally cation selective. This is the first time native function has been demonstrated for recombinant chlamydial MOMP and will have an important impact on the future development of subunit vaccines.
33
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/.
Pełny tekst źródłaStreszczenie:
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.
34
Neary, Tiffany Jonean. "Ultraviolet resonance Raman spectroscopy of the integral membrane protein OmpA elucidating structure and tryptophan microenvironment of folded and unfolded states /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1459291.
Pełny tekst źródłaStreszczenie:
Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed November 13, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references.
35
McGuinness, Brian Timothy. "Immunochemistry and structural variation of the class 1 outer membrane protein of Neisseria meningitidis". Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386663.
Pełny tekst źródła
36
Humes, Julia Rose. "The mechanism of the ATP-independent periplasmic chaperone SurA in outer membrane protein biogenesis". Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/22526/.
Pełny tekst źródłaStreszczenie:
Outer membrane proteins (OMPs) of Gram-negative bacteria travel from their site of synthesis in the cytoplasm, across the inner membrane and through the periplasm to the outer membrane (OM) prior to their folding to a functional form. To protect OMPs from misfolding or aggregation while traversing the periplasm a network of chaperones are employed. The OMPs must then reach the essential β-barrel assembly machinery (BAM) complex, which is involved in inserting OMPs into the OM. This process occurs in the absence of chemical energy as the periplasm is devoid of ATP and in a highly dynamic environment as the 'leaky' OM allows small molecules (< 600 Da) to enter from the extracellular milieu. The major periplasmic chaperone for OMPs, SurA, is known to interact with a number of substrates and has been crosslinked to the BAM complex in vivo. SurA is composed of four domains, an N-terminal domain, two peptidyl-prolyl isomerase (PPIase) domains and a short C-terminal helical domain. In this work wild-type E. coli SurA and SurA truncation variants lacking one (P2) or both (N-Ct) of the PPIase domains have been studied. Using microscale thermophoresis, light scattering, native mass spectrometry and other biophysical techniques how each domain is involved in OMP binding, chaperoning and delivery to BAM is investigated. The results demonstrate that SurA binds unfolded OMPs, tOmpA and OmpT with μM affinity, agreeing with previous findings. The core domain (SurA N-Ct) is sufficient for this interaction, but the addition of the PPIase domains leads to a tighter binding. Light scattering experiments shows that SurA WT can prevent aggregation of the two model OMPs, but the removal of the PPIase domains reduces the chaperoning ability for the larger, more aggregation-prone OMP, OmpT. These observations demonstrate that the acquisition of the PPIase domains is advantageous for both OMP binding and chaperoning. An interaction between SurA and the BAM complex is also observed for the first time in vitro. Overall, the results reveal new insights into how SurA binds and chaperones OMPs before delivering them to the BAM complex for folding in the OM.
37
McMorran, Lindsay Madeline. "Mechanisms of outer membrane protein folding : effects of the lipid environment and periplasmic chaperones". Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/5906/.
Pełny tekst źródłaStreszczenie:
In contrast with the wealth of information on the folding of soluble, cytosolic proteins, little is known about the folding of integral membrane proteins. The outer membrane proteins (OMPs) of Gram-negative bacteria have a β-barrel structure and are essential for cell survival. The mechanisms of OMP transport across the periplasm and how these proteins subsequently fold and insert into the outer membrane remain to be elucidated. The work presented herein examines the folding and membrane insertion of four different OMP constructs. Two homologous bacterial OMPs, OmpT and OmpP, were cloned, over-expressed and purified before biochemical and biophysical methods were employed to examine their folding properties. This work demonstrates that small differences in primary sequence can have large effects on folding efficiency and stability. In spite of both OmpT and OmpP being able to fold under a variety of conditions, it was not possible to establish conditions under which folding was completely reversible. Examination of the origins of irreversible OMP folding was carried out using OmpT, as well as both hexa-histidine tagged and untagged constructs of the outer membrane acyltransferase enzyme, PagP. This study revealed evidence that lipid adhesion of the protein in the unfolded state may be important in preventing aggregation and promoting reversibility. Finally, conditions were established to promote the folding of untagged PagP in low urea concentrations to allow the study of OMPs in the presence of the periplasmic chaperones, SurA and Skp. SurA was shown to have little effect on the folding of PagP into liposomes with zwitterionic or negatively charged membrane surfaces, while Skp was shown to exhibit holdase activity and to modulate PagP folding rate, dependent on the lipid composition. The results present the first detailed insights into the mechanism by which Skp and SurA act to facilitate PagP folding in vitro.
38
Juodeikis, Rokas. "Engineering membranes in Escherichia coli : the magnetosome, LemA protein family and outer membrane vesicles". Thesis, University of Kent, 2016. https://kar.kent.ac.uk/61062/.
Pełny tekst źródłaStreszczenie:
Magnetosomes are membranous organelles found in magnetotactic bacteria (MTB). The organelle consist of ferromagnetic crystals housed within a lipid bilayer chained together by an actin-like filament and allows MTB to orient within magnetic fields. The genetic information required to produce these organelles has been linked to four different operons, encoding for 30 genes. These membranous organelles and the magnetic minerals housed within have various biotechnological applications, therefore enhanced recombinant production of such structures in a model organism holds significant potential. The research described in this thesis is focuses on the production of recombinant magnetosomes in the model organism Escherichia coli. Cloning the genes involved in the generation of the organelle individually or in various combinations resulted in the construction of over 100 different plasmids, compatible with the model organism. SDS-PAGE and electron microscopy analysis was used to characterise E. coli cells harbouring these constructs. The observation of electron dense particles, arranged in a chain structure, show that magnetosome generation in the model organism is possible, but is highly dependent on the growth conditions used. The need for specific growth conditions is later backed up by the analysis of the maturation of the cytochrome c proteins involved in magnetosome biomineralisation, which can only be correctly processed under certain conditions. Individual production of two different magnetosome proteins, MamQ or MamY, allowed the generation of various membranous structures in E. coli observed in 48.9% and 56.2% of the whole population of cells respectively. Combinations of these with MamI, MamL or MamB in a variety of combinations led to a variation in the phenotype observed. Bioinformatics analysis of MamQ led to the discovery of a novel membrane restructuring protein family, the LemA protein family, present in a broad range of bacteria. Four different LemA proteins from Bacillus megaterium, Clostridium kluyveri, Brucella melitensis or Pseudomonas aeruginosa were then produced in E. coli and the analysis of the resulting strains revealed the presence of novel intracellular membranous structures which vary in size, form and localisation. Furthermore, when attempts were made to target these proteins for the modification of the outer membrane, a mechanism for increased outer membrane vesicle generation was serendipitously discovered and different effects of these proteins were once again observed. Together, the results described shows good evidence for recombinant magnetosome production in E. coli and opens a new avenue of membrane engineering in this commonly used organism. Such membranous structures have various biotechnological applications, such as enhanced metabolic engineering potential or specialised lipid vesicle production.
39
au, T. La@murdoch edu, i Tom La. "Characterisation, Recombinant Expression and Immunogenicity of BHLP29.7, An Outer Membrane Lipoprotein of Brachyspira Hyodysenteriae". Murdoch University, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070830.141953.
Pełny tekst źródłaStreszczenie:
Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative 10 and 35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7.
The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system.
An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis.
Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.
40
Rios, Rosa Elvira. "Characterisation of the physiological, chemical and pathogenic changes arising from the adaptation of Campylobacter jejuni to aerotolerant growth". Thesis, Manchester Metropolitan University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243715.
Pełny tekst źródła
41
Fischer, Steven Harold. "Interactions of Neisseria gonorrhoeae with human neutrophils: Gonococcal outer membrane protein II modulates neutrophil responses". Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184364.
Pełny tekst źródłaStreszczenie:
The disease gonorrhea has plagued mankind at least as long as written records have been kept (Black and Sparling, 1985). N. gonorrhoeae is still an important cause of suffering, infertility, and occasional mortality despite the fact that treatment with antibiotics is relatively easy and highly effective, even with the recent increase in penicillin-resistant isolates (Jephcott, 1986). The continued existence of this public health problem is partly the result of a reservoir of asymptomatic carriers within the community who normally don't seek treatment and continue their usual sexual practices (Handsfield, 1983; Kavli et al., 1984). Asymptomatic carriers do not have the purulent discharge characteristic of gonococcal urethritis and cervicitis in which the neutrophil is such a prominent element. Since IgM is present in only trace amounts on genital mucosa (Schumacher, 1973), and this is the "naturally occurring" antibody against gonococci (Rich and Kasper, 1982); it is not unreasonable to assume that non-opsonic chemotaxis and non-opsonic phagocytosis by PMN may play important roles in initiating the inflammatory response and symptomatology seen with gonorrhea. Further, non-opsonic phagocytic killing may be important in eventually clearing gonococcal infection since the role of specific humoral immunity is limited by the ability of gonococcus to constantly vary its antigenic facade (Zak et al., 1984). I have found that three different gonococcal strains express certain outer membrane proteins of the protein II (P.II) family which stimulate neutrophil phagocytic killing and oxidative metabolism in a highly efficient, dose-dependent manner. Other P.IIs expressed by two of the strains are non-stimulatory. Since all P.IIs have very similar physicochemical properties, these results suggest that a specific receptor-ligand interaction occurs between the gonococcal P.II and some element of the neutrophil plasma membrane. The presence or absence of pili on the gonococcal surface has no apparent effect on the ability of certain P.IIs to stimulate neutrophils. Changes in gonococcal outer membrane protein I and lipopolysaccharide, which are thought to confer serum resistance, also have no apparent effect on P.II stimulation of human PMN. Therefore, gonococcal outer membrane P.II may be an important mediator in the inflammatory response to gonococcal infection. Once gonococci are phagocytized by human PMN killing occurs rapidly and there is no evidence of significant intracellular survival. Non-oxidative killing by human chronic granulomatous disease neutrophils is as effective as the killing seen with normal PMN. Extracellular killing of gonococci does not occur to any appreciable extent.
42
Sukumaran, Suja. "Spectroscopic investigation of stability, unfolding and refolding of outer membrane protein porin from Paracoccus denitrificans". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=974953849.
Pełny tekst źródła
43
Bell, Angus. "Structure, function, and role in antibiotic resistance of outer membrane protein H1 in Pseudomonas aeruginosa". Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/29008.
Pełny tekst źródłaStreszczenie:
A divalent cation-regulated outer membrane protein of Pseudomonas aeruginosa, HI, was purified by selective solubilizations of outer membranes in detergent and ethylenediaminetraacetate (EDTA) followed by either two cycles of anion-exchange chromatography or sodium dodecylsulphate-polyacrylamide gel electrophoresis. Protein purified by the former method was contaminated with an equi-molar or higher concentration of lipopolysaccharide , (LPS) that was enriched in 0 side chain-containing molecules, suggesting an association between protein Hi and smooth LPS. Electrophoresis gave higher yields of purified protein Hi, and this product was used for N-terminal amino acid sequencing, amino acid analysis, and polyclonal antiserum production.
Oligodeoxyribonucleotides complementary to the upstream end of the structural gene for protein Hi, oprH, were designed using the N-terminal sequence of the protein. Probing of Southern blots of chromosomal DNA digests with the oligonucleotides revealed that oprH was probably a single-copy gene, and allowed it to be cloned in Escherichia coli. Nucleotide sequence analysis confirmed the cloning of the correct gene, and the derived amino acid sequence indicated a slightly basic protein (in agreement with its proposed function of interacting with anionic sites on LPS) of 178 residues, with two hydrophobic segments.
Protein H1 was produced, proteolytically processed and probably exported to the outer membrane in E. coli cells carrying the complete oprH gene on plasmids. The oprH gene could be expressed weakly from a promoter on the cloned DNA provided that a particular downstream sequence was not deleted. This suggested that the downstream region was involved in regulation of expression of the cloned oprH gene. Protein H1 was produced at levels much higher than background when extra copies of the oprH gene were present in an expression vector in P. aeruginosa. This overproduction of protein H1 caused decreased susceptibility of cells to killing by EDTA, but not by polymyxin B or gentamicin. This partly confirmed the hypothesis (T.I. Nicas and R.E.W. Hancock, J. Bacteriol. 143; 872-878, 1980) that protein HI causes resistance to these agents by inhibiting their self-promoted uptake across the outer membrane. However, an
additional alteration (possibly an increase in cationic
substituents on LPS) was apparently required for the
fully resistant phenotype. This idea was supported by
the observed suppression by LPS mutations of the
polymyxin B resistance of protein H1-overproducing cells,
and by the properties of a P. aeruginosa strain
apparently lacking protein H1.
Several species of bacteria related to P. aeruginosa
produced envelope proteins that were inducible by growth
in Mg²⁺-deficient medium, and one (a protein of apparent
molecular weight 20,000 from P. chloraphis) cross-reacted
immunologically with protein Hi. P. chloraphis, like P.
aeruginosa, was polymyxin B resistant when it was grown
in Mg²⁺-deficient medium.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
44
Berry, Jody Douglas. "Antibody gene markers and their relationship to chlamydial major outer membrane protein (MOMP) immune response". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0006/NQ41602.pdf.
Pełny tekst źródła
45
Butt, Neil James. "The complete nucleotide sequence and immunochemistry of the major outer membrane protein of Neisseria gonorrhoeae". Thesis, University of Southampton, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316059.
Pełny tekst źródła
46
Bulieris, Paula Vasilichia. "Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli". [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-48784.
Pełny tekst źródła
47
Ujwal, Rachma. "Structural and fuctional characterization of the outer mitochondrial membrane protein voltage-dependent anion channel 1/". Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1930281371&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
Pełny tekst źródła
48
Königer, Verena [Verfasser], i Rainer [Akademischer Betreuer] Haas. "CEACAMs as novel receptors for Helicobacter pylori outer membrane protein HopQ / Verena Königer ; Betreuer: Rainer Haas". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1119073731/34.
Pełny tekst źródła
49
Khursigara, Cezar M. "Interactions between the energy-transducing protein TonB and the outer membrane receptor FhuA from Escherichia coli". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85563.
Pełny tekst źródłaStreszczenie:
Iron is an essential element required by most organisms. To acquire iron, Gram-negative bacteria utilize siderophores; compounds that bind iron and form soluble complexes. Escherichia coli imports siderophores via specialized transport systems. A model siderophore receptor system requires the integral outer membrane (OM) receptor FhuA, which facilitates transport of hydroxamate siderophores ferricrocin (Fc) and ferrichrome. Transport of siderophores by FhuA to the periplasm and into the cell requires energy provided by the proton motive force and delivered by the cytoplasmic membrane protein complex TonB-ExbB-ExbD. Energy is transduced from this complex by protein-protein interactions between TonB and FhuA. Although this system has been extensively studied, much remains unclear about interactions between TonB and FhuA.Sedimentation velocity analytical ultracentrifugation (AUC) experiments involving mixtures of recombinant TonB and FhuA were conducted to determine the oligomeric state of TonB-FhuA complexes. These experiments demonstrated that wild-type TonB-FhuA proteins form a 2:1 (TonB:FhuA) complex, and that this complex is more abundant when FhuA is pre-incubated with the siderophore Fc. Deletion of the N-terminal region of TonB also demonstrated a 2:1 complex stoichiometry with FhuA, but in much lower abundance. Deletion of a highly conserved proline-rich region within TonB resulted in the formation of a 1:1 complex, suggesting a novel role for this domain in the formation of a functional 2:1 complex.
Affinities of interaction between wild-type and mutant TonB and FhuA proteins were measured by surface plasmon resonance (SPR). SPR data revealed that wild-type TonB-FhuA interactions are comprised of multiple interaction sites, and undergo an intermediate rearrangement event prior to the formation of the 2:1 complex. Deletions of TonB domains do not disrupt this rearrangement, but do decrease affinity. FhuA cork domain deletions also identified novel sites important for stable interactions.
Information of TonB-FhuA interactions derived by AUC and SPR described herein has contributed greatly to the purification and crystallization of TonB-FhuA co-complexes. Overall, these complementary biophysical techniques have provided important mechanistic details into the protein-mediated transport of iron into Gram-negative bacteria, and will provide a conceptual framework of TonB-FhuA that will help in elucidating aspects of the high-resolution structure of the complex.
50
Malia, Thomas J. 1977. "NMR structural and functional studies of the mithochondrial outer membrane protein VDAC by Thomas J. Malia". Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37889.
Pełny tekst źródłaStreszczenie:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006."September 2006." Vita.
Includes bibliographical references.
Apoptosis is a mechanism of programmed cell death required by multicellular organisms during development and for tissue maintenance. Bcl-2 family proteins are central regulators of apoptosis and many of their primary roles are carried out in the outer mitochondrial membrane. Anti-apoptotic Bcl-xL has been found previously to interact with VDAC, an outer mitochondrial membrane protein responsible for metabolite trafficking. Here, the NMR-based investigation of VDAC and its interaction with Bcl-xL in detergent micelles is described. As an integral membrane protein VDAC presented a challenge for producing a folded form amenable to solution NMR studies. Methods developed for expression and purification of human VDAC for structural studies by NMR spectroscopy were developed. Optimal sample conditions were explored in a screen of various detergents and buffers. Suitable NMR sample conditions for VDAC were identified and allowed further characterization of VDAC and its interactions by NMR and other methods. Obtained through various means, evidence of a concentration dependent self-association of VDAC is also presented. Chemical cross-linking, analytical size exclusion chromatography, and NMR spectroscopic studies strongly support an equilibrium model for LDAO-purified VDAC between monomer and trimer.
(cont.) Methods that were necessary to carry out NMR structural studies of VDAC and results from those studies are described. In addition to various methods previously developed for solution NMR spectroscopy of very large proteins, extensive labeling approaches and unconventional experimental NMR methods were necessary to obtain a high level of chemical shift assignment for micelle-bound VDAC. Chemical shift assignment has allowed the preliminary characterization of ATP and Bcl-xL binding to VDAC. Binding of Bcl-xL, a central regulator of apoptosis, to VDAC is demonstrated here. Using NMR spectroscopy, the VDAC-binding region of Bcl-xL has been mapped to a putative helical hairpin motif, which also mediates insertion into membranes. The stoichiometry of the VDAC/Bcl-xL complex is shown to be a heterotrimer of two VDAC monomers and one Bcl-xL. The demonstration of VDAC/Bcl-xL complex formation supports the concept that components of apoptosis and metabolism are integrally connected, and that interplay between the two processes is required for regulation of cell survival and cell death.
Ph.D.