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Engineering peptide specific hyper-crystallizable antibody fragments (scFv) as potential chaperones for co-crystallizationPai, Jennifer Chentzu 09 February 2011 (has links)
Hydrophobic membrane proteins perform a variety of important functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. We aim to develop a toolbox of peptide specific single-chain antibody fragment chaperones engineered for hyper-crystallizability. These peptide sequences can be introduced into various regions of membrane proteins without interfering with protein function. The resulting protein-chaperone complex is expected to form a crystal lattice mediated by chaperone interactions.
We have developed candidate scFv chaperone proteins binding hexa-histidine (His6) and EYMPME (EE) tags with improved biophysical features influencing crystallization propensity, including peptide affinity, stability and solubility. The scFv libraries were generated using a novel ligation-free technique, MegAnneal, allowing us to rapidly generate large libraries based on 3D5 scFv. We identified two candidate chaperones, 3D5/His_683, specific for His6 and 3D5/EE_48, specific for EE tags. Variants exhibit high solubility (up to 16.6 mg/ml) and nanomolar peptide affinities; complexes of 3D5/EE_48 with EE-tagged proteins were isolated by gel filtration. We have developed design rules for EE peptide placement at terminal, inter-domain or internal loop regions of the target protein to balance peptide accessibility for chaperone binding while retaining rigid protein-chaperone complexes suitable for crystallization.
The 3D5/ His_683 crystallized in four different conditions, utilizing multiple space groups. The 3D5/EE_48 scFv was crystallized (3.1 Å), revealing a ~52 Å channel in the crystal lattice, which may accommodate a small peptide-tagged target protein. Our evolution experiments altered scFv surface residues, resulting in use of different crystallization contacts. Analysis of these crystal contacts and those used by crystallized 14B7 scFv variants, led us to postulate that lattice formation is driven by strong crystal contacts. To test this hypothesis, we introduced amino acid changes expected to reduce the affinity of the 3D5/EE_48 energetically dominant crystal contacts. This approach to crystal contact engineering may allow semi-rational control over lattice networks preferred by scFv chaperones. Co-crystallization trials with model proteins are on-going. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries. / text
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The Clathrin Adaptor AP-1 and Type II Phosphatidylinositol 4-Kinase are Required for Glue Granule Biogenesis in DrosophilaBurgess, Jason 06 December 2012 (has links)
Regulated secretion of hormones, digestive enzymes and other biologically active molecules requires formation of secretory granules. However, the molecular machinery required for secretory granule biogenesis is incompletely understood. I used powerful genetic approaches available in the fruit fly Drosophila melanogaster to investigate the factors required for biogenesis of mucin-containing ‘glue granules,’ which form within epithelial cells of the third-instar larval salivary gland. I discovered that clathrin and the clathrin adaptor protein complex (AP-1), as well the enzyme type II phosphatidylinositol 4-kinase (PI4KII), are indispensable for glue granule biogenesis.
Clathrin and AP-1 are necessary for maturation of exocrine, endocrine and neuroendocrine secretory granules in mammalian cells. I found that Drosophila clathrin and AP-1 colocalize at the TGN and that clathrin recruitment requires AP-1. I further showed that clathrin and AP-1 colocalize with secretory cargo at the TGN and on glue granules. Finally, I demonstrated that loss of clathrin or AP-1 leads to a profound block in secretory granule biogenesis. These findings establish a novel role for AP-1/clathrin-dependent trafficking in the formation of mucin-containing secretory granules.
Type II phosphatidylinositol 4-kinase (PI4KII) generates the membrane lipid phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network and is required to recruit cargo to endosomes in mammalian cells. I generated null mutations in the sole Drosophila PI4KII and demonstrated a role for PI4KII in both glue granule and pigment granule biogenesis. PI4KII mutant salivary gland cells exhibit small glue granules and mislocalize glue protein to abnormally large late endosomes. Additionally, PI4KII mutants exhibit altered distribution of the granule specific SNARE, SNAP-24. These data point to a crucial role for PI4KII in sorting of regulated secretory products during granule biogenesis. Together, my results indicate that the larval salivary gland is a valuable system for investigating molecular mechanisms involved in secretory granule biogenesis, and provide a framework for future studies using this system.
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Global Proteomic Detection of Native, Stable, Soluble Human Protein ComplexesHavugimana, Pierre Claver 12 December 2012 (has links)
Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably-associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis-driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution.
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Analyses of the proteins KpsM, KpsE and KpsD in the group 2 capsular polysaccharide export complex of Escherichia coliHaas, Eva January 2012 (has links)
The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.
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Structural and functional studies of mitochondrial small Tim proteinsGuo, Liang January 2013 (has links)
Most mitochondrial proteins are encoded by nuclear DNA, and synthesised in the cytosol, then imported into the different mitochondrial subcompartments. To reach their destination, mitochondrial inner membrane proteins require import across the outer mitochondrial membrane, and through the intermembrane space. This passage through the IMS is assisted by the small Tim proteins. This family is characterised by conserved cysteine residues arranged in a twin CX3C motif. They can form Tim9-Tim10 and Tim8-Tim13 complexes, while Tim12 appears to form part of a Tim9-Tim10-Tim12 complex that is associated with the inner membrane translocase TIM22 complex. Current models suggest that the biogenesis of small Tim proteins and their assembly into complexes is dependent on the redox states of the proteins. However, the role of the conserved cysteine residues, and the disulphide bonds formed by them, in small Tim biogenesis and complex formation is not clear. As there is no research about the structural characterisation of Tim12 and double cysteine mutants of Tim9, purification of these proteins was attempted using different methods. To investigate how cysteine mutants affect complex formation, the purified double cysteine mutants of Tim9 were studied using in vitro methods. It showed that the double cysteine mutants were partially folded, and they can form complexes with Tim10 with low affinities, suggesting disulphide bonds are important for the structures and complex formation of small Tim proteins. The effect of cysteine mutants on mitochondrial function was addressed using in vivo methods. It showed that cysteines of small Tim proteins were not equally essential for cell viability, and growth defect of the lethal cysteine mutant was caused by low level of protein. Thus, the conclusion of this study is that disulphide bond formation is highly important for correct Tim9- Tim10 complex formation, and yeast can survive with low levels of complex, but it results in instability of the individual proteins.
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Characterization of RNA and RNA-Protein Complexes by Native Mass SpectrometrySarni, Samantha H. January 2020 (has links)
No description available.
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Polyphenols: Interactions with proteins and analytical methodsTrombley, John D. 05 December 2011 (has links)
No description available.
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Characterization of TaXPol-1, a Xylan Synthase Complex from WheatJiang, Nan 17 September 2015 (has links)
No description available.
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The regulation of Atg1 protein kinase activity is important to the autophagy process in <i>Saccharomyces cerevisiae</i>Yeh, Yuh-Ying 15 December 2010 (has links)
No description available.
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THE ROLE OF AMPK IN THE EXPRESSION OF THE DAPC / THE ROLE OF AMPK IN THE EXPRESSION OF THE DYSTROPHIN-ASSOCIATED PROTEIN COMPLEX IN SKELETAL MUSCLEDial, Athan January 2017 (has links)
The dystrophin-associated protein complex (DAPC) provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, serving as a mechanosensor and signal transducer across the sarcolemma. Pharmacological stimulation of AMP-activated protein kinase (AMPK) induces the expression of DAPC components in skeletal muscle, whereas physiological reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast glycolytic extensor digitorum longus (EDL) and slow oxidative soleus (SOL) muscles from wild-type (WT) mice, as well as from littermates deficient in both isoforms of the AMPK-β subunit in skeletal muscle (MKO) were analyzed. DAPC mRNA levels, as well as protein expression and localization were similar between genotypes, with the exception of nNOS, which displayed a compensatory sarcolemmal enrichment in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC, such as PGC-1α and KSRP, were also not affected by the loss of AMPK. However, MyoD and myogenin expression was significantly diminished in MKO muscles, which is consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO SOL muscles, despite an adaptive accumulation of PGC-1α at the sarcolemmal compartment. Collectively the evidence indicates that AMPK is sufficient, but not essential for the maintenance of DAPC expression in skeletal muscle. However, AMPK is required for preserving extrasynaptic utrophin levels in slow, oxidative muscles, which underscores the role of AMPK in the gene expression of this disease modifying protein. / Thesis / Master of Science (MSc) / The dystrophin-associated protein complex (DAPC) connects the interior and exterior of muscle cells. Activation of AMP-activated protein kinase (AMPK) increases the expression of the DAPC in skeletal muscle. We sought to determine whether AMPK was necessary for DAPC expression in skeletal muscle. Fast and slow muscles from normal mice, as well as from those deficient in skeletal muscle AMPK (MKO) were analyzed. We found DAPC levels and localization were similar between both groups, with the exception of nNOS, which was enriched at the muscle membrane in MKO muscles. Regulators of the DAPC were also not affected by the loss of AMPK. However, genes important for the production of muscle were significantly diminished in MKO muscles. Furthermore, we observed decrements in utrophin at the muscle membrane selectively in slow MKO muscles. Our work indicates that AMPK is not essential for the DAPC expression in skeletal muscle, however it is required for preserving utrophin levels in slow, oxidative muscles.
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