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20S proteasome assembly: alternative pathways and complexesHammack, Lindsay J. January 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The ubiquitin-proteasome system is responsible for the targeted degradation of proteins within the cell. The 26S proteasome, which is the protease of this system, is a high molecular weight complex consisting of 33 subunits that arrange to form two smaller complexes the 19S regulatory particle (RP) and the 20S core particle (CP). The 19S RP can bind one or both ends of the 20S CP and is responsible for recognizing the ubiquitinated substrates. After recognition, the 19S RP will subsequently deubiquitinate, unfold, and translocate the substrates into the proteolytic 20S CP. The 20S CP consists of seven unique alpha and seven unique beta subunits that arrange into four stacked rings, with two alpha rings capping two beta rings. Assembly of the alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) structure begins with the formation of an alpha ring and proceeds through specific assembly intermediates. This process is assisted by assembly chaperone proteins that promote on pathway interactions to efficiently construct the 20S CP. In this dissertation, three new findings are described which further characterize the proteasome assembly pathway. First, novel non-canonical complexes comprised of proteasome subunit alpha4 were identified in vivo, revealing proteasome subunits can assemble into complexes outside of the proteasome. Second, Hsp70 proteins, Ssa1/2, were shown to assist in the assembly of 20S CPs, adding to the growing list of proteins guiding proteasome assembly. Third, a novel complex was identified which is believed to represent a new proteasome assembly intermediate.
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BIOCHEMICAL CHARACTERIZATION OF ADIPONECTIN OLIGOMERIZATIONBriggs, David Blaine January 2011 (has links)
Adiponectin, a hormone that homo-oligomerizes into trimer, hexamer, or higher molecular weight (HMW) species, is involved in maintaining insulin sensitivity in muscle and liver. Interestingly, its functions appear to be oligomer-specific. Recent data suggest that HMW levels are decreased in obesity and insulin resistance, although, the cause for this decrease is not known. Impaired assembly to the octadecamer represents one possible reason for decreased HMW adiponectin in insulin resistance and type 2 diabetes, but mechanisms by which HMW adiponectin assembles are unknown. This dissertation discusses the progress that we have made regarding formation of HMW adiponectin in vitro.I found that disulfide bonds are important in the assembly process to octadecameric adiponectin, but are not required for stability of the octadecamer itself. We showed that hydrogen peroxide accelerated oligomerization to the octadecamer through formation of disulfide bonds, while alkylation of the cysteines led to inhibition of both oligomerization and disulfide bond formation. Using comparative native/denaturing polyacrylamide gel electrophoresis (PAGE), dynamic light scattering, and tandem mass spectrometry, we demonstrated that octadecamer is stable in the absence of disulfide bonds by using multiple biochemical and biophysical assays. In addition, oxidized adiponectin oligomerizes to octadecamer far slower than reduced adiponectin. To further evaluate the role of disulfide bonds in the formation to octadecamer, we analyzed the role of reduction potential on adiponectin oligomerization. We observed that under immediate oxidizing conditions, hexamers and trimers form. Oxidized hexamer can form HMW adiponectin through disulfide bond rearrangement using beta-mercaptoethanol (βME) or increasing the total concentration of glutathione under oxidizing conditions. To further understand the role of disulfide bonds, we showed that zinc increased the oligomerization to octadecamer. This effect was associated with decreased initial disulfide bonding during the assembly to the octadecamer. In summary, these data suggest the rate of disulfide bond formation and the ability to undergo disulfide bond isomerization are important in the oligomerization process of HMW adiponectin.
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Characterization of an Iron-Sulfur Binding Protein in the Tail Tip Complex of Bacteriophage LambdaTam, William 27 November 2013 (has links)
The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.
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Characterization of an Iron-Sulfur Binding Protein in the Tail Tip Complex of Bacteriophage LambdaTam, William 27 November 2013 (has links)
The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.
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Characterizing the Structure and Mechanics of 2D Clathrin Lattices with Atomic Force MicroscopyPlaten, Mitja 22 October 2015 (has links)
No description available.
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Studying Molecular Interactions under Flow with Fluorescence Fluctuation SpectroscopyPerego, Eleonora 16 January 2019 (has links)
No description available.
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Rational Metalloprotein Design for Energy Conversion ApplicationsJanuary 2019 (has links)
abstract: Continuing and increasing reliance on fossil fuels to satisfy our population’s energy demands has encouraged the search for renewable carbon-free and carbon-neutral sources, such as hydrogen gas or CO2 reduction products. Inspired by nature, one of the objectives of this dissertation was to develop protein-based strategies that can be applied in the production of green fuels. The first project of this dissertation aimed at developing a controllable strategy to incorporate domains with different functions (e. g. catalytic sites, electron transfer modules, light absorbing subunits) into a single multicomponent system. This was accomplished through the rational design of 2,2’-bipyridine modified dimeric peptides that allowed their metal-directed oligomerization by forming tris(bipyridine) complexes, thus resulting in the formation of a hexameric assembly.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019
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Consistent Fabrication of Ultrasmall PLGA Nanoparticles and their Potential Biomedical ApplicationsLohneis, Taylor Paige 04 December 2019 (has links)
Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering.
The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM.
By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation. / Master of Science / Nanotechnology, the manipulation of materials on an atomic or molecular scale, and its potential for biomedical applications has become an area of increasing interest over the last few decades. Nanoparticles, spherical or non-spherical entities of sizes approximately one-billionth of a meter, have been used to solve a wide variety of biomedical problems. For reference, a human hair is about 80,000 to 100,000 nm in size and the nanoscale typically ranges in size from 1 to 1000 nm. This size range is not visible to the naked eye, so methods of analysis via scientific equipment becomes paramount. Specifically, this study aims to fabricate ultrasmall nanoparticles, ranging in size from 5 to 50 nm, which are highly sought after for their physical and chemical properties and their ability to easily travel though the bloodstream. By adjusting the material properties, size, shape, surface charge, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug delivery, detection of viruses, and tissue engineering.
The specific aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs), a type of polymer, using a quick and easy nanoprecipitation method1, with some modifications. Nanoprecipitation occurs by combining two liquid solutions – PLGA and aqueous poly(vinyl alcohol) (PVA) – which interact chemically to form a solid component – a polymer nanoparticle. These two solutions, at varying concentrations, produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. Data collected from this study can be used to select a desired nanoparticle size given a potential application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions.
By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs can be used for a variety of biomedical applications depending on the goal of the furthered study. Two PLGA NP example applications are tested for in this work – in DNA loading and in encapsulation of virus-like particles (VLPs), which are synthetically produced proteins that can be neatly folded to resemble a virus. These VLPs can be used to as an alternative to live vaccines and they can be designed to stimulate the immune system. Positive initial results from this study confirm the potential of these nanoparticles to have a wide impact on the biomedical field depending on specific tailoring to a given application.
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Molecular chaperones in the assembly of α-Synuclein and Parkinson’s Disease / Les chaperons moléculaires dans l’assemblage de l’α-Synucléine et la maladie de ParkinsonPemberton, Samantha 09 December 2011 (has links)
La formation et le dépôt de fibres d'α-Synucléine dans le cerveau humain sont à l‟origine de la maladie de Parkinson. Cette thèse documente le rôle de deux chaperons moléculaires dans l‟assemblage en fibres de l'α-Syn : Hsc70 (protéine de choc thermique constitutivement exprimée chez l‟Homme) et Ssa1p (son équivalent chez la levure). Le but était d'élargir le catalogue d'effets connus des chaperons moléculaires sur α-Syn, pour éventuellement ouvrir la voie à des applications thérapeutiques. Nous avons montré que Hsc70 inhibe l'assemblage de l'α-Syn en fibres, en se liant avec une forte affinité à la forme soluble de l'α-Syn. Hsc70 se lie préférentiellement aux fibres de l'α-Syn, et cette liaison a un effet cytoprotecteur puisqu'elle rend les fibres moins toxiques pour les cellules de mammifères en culture. Pareillement à Hsc70, Ssa1p inhibe l'assemblage de l'α-Syn en fibres, et a une plus forte affinité pour les fibres que pour la forme soluble de l'α-Syn. En revanche, la liaison de Ssa1p aux fibres de l'α-Syn n'a pas d'effet cytoprotecteur, sûrement due aux différences entre les séquences du site de liaison aux peptides des deux chaperons moléculaires, qui fait que Ssa1p a une affinité plus faible que Hsc70 pour les fibres d'α-Syn. Nous avons fixé le complexe entre Ssa1p et α-Syn avec des agents pontants, pour ensuite établir une carte du site d'interaction entre les deux protéines en utilisant la spectrométrie de masse. Ceci est indispensable si un « mini » Ssa1p, constitué des éléments nécessaires et suffisants sera utilisé comme agent thérapeutique pour réduire la toxicité des fibres d'α-Syn. / The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson’s disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a “mini” Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.
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Studium poruch cytochrom c oxidasy a ATP synthasy na biochemické a molekulární úrovni / Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficienciesFornůsková, Daniela January 2011 (has links)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
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