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The novel mouse [gamma]A-crystallin mutation leads to misfolded protein aggregate and cataractCheng, Man-hei. January 2009 (has links)
Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-115). Also available in print.
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Free energy functions in protein structural stability and folding kinetics /Morozov, Alexandre V., January 2003 (has links)
Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 96-115).
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Improving the prediction of RNA secondary structure and automatic alignment of RNa sequencesGardner, David Paul 02 July 2012 (has links)
The accurate prediction of an RNA secondary structure from its sequence will enhance the experimental design and interpretation for the increasing number of scientists that study RNA. While the computer programs that make these predictions have improved, additional improvements are necessary, in particular for larger RNAs. The first major section of this dissertation is concerned with improving the prediction accuracy of RNA secondary structures by generating new energetic parameters and evaluating a new RNA folding model. Statistical potentials for hairpin and internal loops produce significantly higher prediction accuracy when compared with nine other folding programs. While more improvements can be made to the energetic parameters used by secondary structure folding programs, I believe that a new approach is also necessary. I describe a RNA folding model that is predicated on a large body of computational and experimental work. This model includes energetics, contact distance, competition and a folding pathway. Each component of this folding model is evaluated and substantiated for its validity.
The statistical potentials were created with comparative analysis. Comparative analysis requires the creation of highly accurate multiple RNA sequence alignments. The second major section of this dissertation is focused on my template-based sequence aligner, CRWAlign. Multiple sequence aligners generally run into problems when the pairwise sequence identity drops too low. By utilizing multiple dimensions of data to establish a profile for each position in a template alignment, CRWAlign is able to align new sequences with high accuracy even for pairs of sequence with low identity. / text
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Local and global investigations into DEAD-box protein functionPotratz, Jeffrey Philip 13 November 2013 (has links)
Numerous essential cellular processes, such as gene regulation and tRNA processing, are carried out by structured RNAs. While in vitro most RNAs become kinetically trapped in non-functional misfolded states that render them inactive on a biologically-relevant time scale, RNAs folding in vivo do not share this same outcome. RNAs do indeed misfold in the cell; however, chaperone proteins promote escape from these non-native states and foster folding to functional conformations. DEAD-box proteins are ATP-dependent RNA chaperone proteins that function by disrupting structure, which can facilitate structural conversions. Here, studies with both local and global focuses are used to uncover mechanistic features of DEAD-box proteins CYT-19 and Mss116p. Both of these proteins are general RNA chaperones as they each have the ability to facilitate proper folding of multiple structured RNAs. The first study probes how DEAD-box proteins interact with a simple duplex substrate. Separating the strands of a duplex is an ATP-dependent process and is central to structural disruption by DEAD-box proteins. Here, how ATP is utilized during duplex separation is monitored by comparing ATP hydrolysis rates with strand separation rates. Results indicate that one ATP molecule is sufficient for complete separation of a 6-11 base pair RNA duplex. Under some conditions, ATP binding in the absence of hydrolysis is sufficient for duplex separation. Next, focus is shifted to a more global perspective as the function of Mss116p is probed in the folding of a cognate group II intron substrate, aI5[gamma], under near-physiological conditions. Three catalytically-active constructs of aI5[gamma] are used and catalysis serves as a proxy for folding. Folding of all constructs is promoted by the presence of Mss116p and ATP. In vitro and in vivo results indicate that a local unfolding event is promoted by Mss116p, stimulating formation of the native state. Lastly, orthogonal methods that probe physical features of RNA are used to provide insight into the structural intermediates with which Mss116p acts. / text
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Mechanistic studies of the RNA chaperone activities of the DEAD-box RNA helicase CYT-19Jarmoskaite, Inga 07 July 2014 (has links)
Structured RNAs are pervasive in biology, spanning a functional repertoire that includes messengers, regulators of gene expression and catalysts of translation and splicing. From the relatively simple tRNAs and riboswitches to the highly structured ribosomal RNAs, the ability of RNAs to function is dependent on well-defined secondary and tertiary structures. However, studies of RNA folding in vitro have revealed an extreme propensity to form alternative structures, which can be long-lived and interfere with function. In the cell, a diverse array of RNA binding proteins and RNA chaperones guide RNAs towards the correct structure and disrupt misfolded intermediates. Among these proteins, DEAD-box protein family stands out as one of the largest groups, with its members ubiquitously involved in RNA metabolism across all domains of life. DEAD-box proteins can function as both specific and general RNA chaperones by disrupting RNA structures in an ATP-dependent manner. Here I describe my work studying the general RNA chaperone mechanism of the Neurospora crassa protein CYT-19, a model DEAD-box protein and a biological RNA chaperone that is required for efficient folding of self-splicing group I intron RNAs in vivo. After an introduction to DEAD-box proteins and their mechanisms as RNA remodelers (Chapter 1), I will first describe studies of group I intron unfolding by CYT-19, focusing on the effects of RNA tertiary structure stability on CYT-19 activity and targeting to RNA substrates (Chapter 2). I will then describe the characterization of ATP-dependent mechanisms during CYT-19-mediated refolding of the misfolded group I intron (Chapter 3). In Chapter 4, I will present small-angle X-ray scattering (SAXS) studies of structural features of DEAD-box proteins that allow them to efficiently interact with large structured RNA substrates. Finally, I will turn to studies of DEAD-box protein involvement during early steps of RNA compaction and folding, using SAXS and activity-based approaches (Chapter 5). I will conclude with a general discussion of superfamily 2 RNA helicases, which include DEAD-box and related proteins, and their functions and mechanisms as remodelers of structured RNAs and RNPs. / text
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A study of biological role of reactive oxygen species in cellular response in stressLam, Dennis, 林勁行 January 2012 (has links)
When proteins are unable to fold properly in the endoplasmic reticulum (ER), the
resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress
often have a higher abundance of reactive oxygen species (ROS). Previous studies
suggest that ROS could aggravate ER stress by further disrupting the ER protein
folding process. More recent studies suggest that the unfolded protein response
signaling pathways activated by ER stress could lead to the production of ROS. Such
studies lead to the hypothesis that ER stress could be promoted by ROS, and vice
versa. The aim of the present study is to test the above hypothesis by studying how
ROS could be generated in ER-stressed cells. This is followed by investigating if ROS
could increase or decrease the level of ER stress in cells. Finally, the extent of ER
stress induced cell death in the presence and absence of ROS is assessed.
The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress
inducing agent, resulted in the elevation of intracellular ROS that could be detected
with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not
dihydroethidium which is relatively specific towards superoxide anion. The
Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol
reductants such as dithiothreitol and N-acetylcysteine but not with the free radical
scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be
prevented by the over-expression of catalase in HeLa. These data is consistent with
the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells.
In addition to elevation of ROS level, HeLa cells treated with tunicamycin also
resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the
presence of cycloheximide to inhibit protein synthesis so as to deplete protein
substrates for folding in the ER, tunicamycin-induced ER stress was greatly
minimized as was evident by the absence of both the phosphorylation of PERK and
splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of
DCF-detectable ROS remained unaffected. The cycloheximde-resistant
phosphorylation of eIF2α could be prevented when cells were co-treated with thiol
reductants, or upon the over-expression of catalase. These data suggest that the
production of ROS in Tm-treated cells does not require the presence of ER stress as a
prerequisite. Furthermore, the ROS so produced could induce phosphorylation of
eIF2α without the need to cause ER stress in the first place.
The quenching of ROS through the use of thiol reductants, or the over-expression
of catalase, had no effect on inhibition of protein synthesis in cells treated with
tunicamycin. However, the extent of cell death was significantly increased. The data
obtained in this study is not consistent with the idea that ROS is a downstream
product of ER stress, capable of inducing more ER-stress by a feedback mechanism.
Therefore, a mutually enhancing effect between ER stress and ROS may not exist.
The ROS found in stressed cells may serve to extend cellular survival under the
condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
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Exploiting aromatic donor-acceptor recognition in the folding and binding of naphthyl oligomersGabriel, Gregory John 28 August 2008 (has links)
Not available / text
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The engineering of de novo pathways for oxidative protein folding in Escherichia coliMasip, Lluis 28 August 2008 (has links)
Not available / text
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Coarse-grained modeling of concentrated protein solutionsCheung, Jason Ka Jen 28 August 2008 (has links)
Not available / text
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Dynamics of peptide chains during co-translational translocation, membrane integration & domain foldingHedman, Rickard January 2015 (has links)
The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts. The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.
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