Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 124-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 25, 2009). Includes bibliographical references.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-143).

The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes. This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions. The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured. / QC 20100826

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 219-242). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.