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The Role of N- and C-terminal Amino Acids to Prosegment Catalyzed Folding in Porcine Pepsinogen AMyers, Brenna 09 May 2012 (has links)
This thesis is an investigation of the role of the prosegment (PS) of pepsinogen in the binding, refolding and inhibition of pepsin. Native pepsin (Np) is irreversibly denatured, and folds to a stable, non-native state under refolding conditions, termed refolded pepsin (Rp) (Dee and Yada 2010). When added separately, the PS binds Rp, catalyzes folding to the native-like state and inhibits Np (Dee and Yada 2010). It was hypothesized, owing to the high sequence conservation, that N-terminal PS residues are critical to PS catalyzed folding. Synthetic peptides of N-terminal truncations (N16, N29), C-terminal truncations (C15, C28), and full length, wild-type (Wt) PS were examined. N-terminal residues were required for binding to Rp and catalyzing folding, while both N29 and C28 truncations had similar inhibition constants. Remarkably, the foldase activity of N-terminal truncation (N29) was only 2.5 fold slower than Wt, supporting that PS foldase activity is stored almost entirely within the highly conserved N29 region.
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Parallelizable manifold compactifications of D=11 SupergravityGoranci, Roberto January 2016 (has links)
In this thesis we present solutions of spontaneous compactifications of D=11, N=1 supergravity on parallelizable manifolds S^1, S^3 and S^7. In Freund-Rubin compactifications one usually obtains AdS vacua in 4D, these solutions usually sets the fermionic VEV's to zero. However giving them non zero VEV's allows us to define torsion given by the fermionic bilinears that essentially flattens the geometry giving us a vanishing cosmological constant on M_4. We further give an analysis of the consistent truncation of the bosonic sector of D=11 supergravity on a S^3 manifold and relate this to other known consistent truncation compactifications. We also consider the squashed S^7 where we check for surviving supersymmetries by analyzing the generalised holonomy, this compactification is of interest in phenomenology.
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Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivatives / Bioengineering von S-layern: Molekulare Charakterisierung eines neuen S-layer Gens sslA aus Sporosarcina ureae ATCC 13881 sowie nanotechnologische Anwendung von SslA-Protein DerivatenRyzhkov, Pavel 27 February 2008 (has links) (PDF)
S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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Two dimensional Maximal Supergravity, Consistent Truncations and HolographyOrtiz, Thomas 07 July 2014 (has links) (PDF)
A complete non trivial supersymmetric deformation of the maximal supergravity in two dimensions is achieved by the gauging of a SO(9) group. The resulting theory describes the reduction of type IIA supergravity on an AdS_2 x S^8 background and is of first importance in the Domain-Wall / Quantum Field theory correspondence for the D0-brane case. To prepare the construction of the SO(9) gauged maximal supergravity, we focus on the eleven dimensional supergravity and the maximal supergravity in three dimensions since they give rise to important off-shell inequivalent formulations of the ungauged theory in two dimensions. The embedding tensor formalism is presented, allowing for a general desciption of the gaugings consistent with supersymmetry. The SO(9) supergravity is explicitly constructed and applications are considered. In particular, an embedding of the bosonic sector of the two-dimensional theory into type IIA supergravity is obtained. Hence, the Cartan truncation of the SO(9) supergravity is proved to be consistent. This motivated holographic applications. Therefore, correlation functions for operators in dual Matrix models are derived from the study of gravity side excitations around half BPS backgrounds. These results are fully discussed and outlooks are presented.
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Two dimensional Maximal Supergravity, Consistent Truncations and Holography / Supergravité maximale bidimensionnelle, troncatures cohérentes et holographieOrtiz, Thomas 07 July 2014 (has links)
Nous avons réalisé une déformation non-triviale et complète de la théorie de supergravité maximale en dimension deux. Il s'agit de la supergravité maximale avec groupe de jauge SO(9). Cette théorie décrit de manière effective la supergravité de type IIA sur un espace-temps produit AdS_2 x S^8. Elle joue ainsi un rôle important dans la correspondance Gravité / Théorie de Jauge appliquée au cas de la D0-brane. Afin de préparer la construction de la supergravité maximale jaugée SO(9), nous nous intéressons aux supergravités maximales en dimension onze et trois, puisqu'elles donnent lieu à différentes formulations non équivalentes de la théorie bidimensionnelle non jaugée. Le formalisme d' « Embedding tensor » est ensuite présenté. Il permet de déterminer l'ensemble des groupes de jauges compatibles avec la supersymétrie maximale. La supergravité SO(9) est dès lors explicitement construite et ouvre la voie à deux applications importantes. P our commencer, nous avons réalisé l'inclusion d'un sous-secteur bosonique de la théorie SO(9), la troncature de Cartan, dans la supergravité de type IIA à dix dimensions d'espace-temps. Il s'agit d'une inclusion cohérente. Cela a motivé la deuxième application, de nature holographique. Ainsi, à partir du sous-secteur de Cartan de la supergravité SO(9), et en particulier de la découverte d'états fondamentaux de type « half-BPS », nous avons calculé un ensemble de fonctions de corrélation à un et deux points associées à des opérateurs de modèles de matrice duaux. Nous avons conclu en un résumé de nos travaux et en la présentation d'intéressantes perspectives. / A complete non trivial supersymmetric deformation of the maximal supergravity in two dimensions is achieved by the gauging of a SO(9) group. The resulting theory describes the reduction of type IIA supergravity on an AdS_2 x S^8 background and is of first importance in the Domain-Wall / Quantum Field theory correspondence for the D0-brane case. To prepare the construction of the SO(9) gauged maximal supergravity, we focus on the eleven dimensional supergravity and the maximal supergravity in three dimensions since they give rise to important off-shell inequivalent formulations of the ungauged theory in two dimensions. The embedding tensor formalism is presented, allowing for a general desciption of the gaugings consistent with supersymmetry. The SO(9) supergravity is explicitly constructed and applications are considered. In particular, an embedding of the bosonic sector of the two-dimensional theory into type IIA supergravity is obtained. Hence, the Cartan truncation of the SO(9) supergravity is proved to be consistent. This motivated holographic applications. Therefore, correlation functions for operators in dual Matrix models are derived from the study of gravity side excitations around half BPS backgrounds. These results are fully discussed and outlooks are presented.
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Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivativesRyzhkov, Pavel 17 October 2007 (has links)
S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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