Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 142 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Abraham M. Lenhoff, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (M.S.)--Humboldt State University, 2007. / Includes bibliographical references (leaves 81-82). Also available via Humboldt Digital Scholar.

Thesis (Ph. D.)--University of Delaware, 2006. / Principal faculty advisor: Brian J. Bahnson, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

No description available.

abstract: Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles. / Dissertation/Thesis / Ph.D. Microbiology 2010

The rhomboid family of intramembrane serine proteases can catalyze proteolysis of substrates that are normally embedded in the cell membrane, making them key players in a diverse range of biological processes. While X-ray crystal structures provide detailed insights into the mechanism of intramembrane hydrolysis, questions remain concerning how transmembrane (TM) substrates are able to gain access to the rhomboid active site, and whether interactions with the membrane environment can influence its structure and function. In this thesis, these questions were investigated using the E. coli rhomboid ecGlpG. In Chapter 3, the effect of hydrophobic mismatch between lipid and protein was investigated using families of amphiphiles with saturated alkyl chains. While ecGlpG displayed maximal activity against a water-soluble model substrate when solubilized in detergents containing 10-12 carbon atoms, shorter and longer chain detergents led to loss of activity. An even larger effect was observed when ecGlpG was reconstituted into phospholipid bicelles, with no proteolytic activity being detected in 14-carbon lipids. These results suggest that mismatch between the hydrophobic regions of the catalytic TM domain (TMD) and the local membrane environment is detrimental to proteolysis. To obtain further insight into the structure and dynamics of ecGlpG, sample conditions were identified in Chapter 4 that enabled, for the first time, the acquisition of NMR spectra showing signals from the ecGlpG TMD. While significant peak broadening prevented chemical shift assignment, the sensitivity and resolution of peaks corresponding to the tryptophan indole NH group allowed their use as structural probes. These were employed in Chapter 5 to characterize the open conformation of ecGlpG that is postulated to facilitate substrate entry. These spectra showed evidence of an open conformation in which the intact α5 is laterally displaced. Interactions with a substrate-derived peptide also appeared to stimulate gate opening; however, activity assays suggested that formation of the open state could compromise catalytic activity against water-soluble substrates, and that interactions with TM substrates could counter this effect. Taken together, these results provide new insight into the role of both the local membrane environment and α5-conformational dynamics on intramembrane proteolysis, and suggest a mechanism to prevent cleavage of off-target rhomboid substrates in vivo.

Li, Kwun Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 83-88). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory and Endocytic Pathways --- p.2 / Chapter 1.2 --- PVC Proteomics Analysis Led to the Identification of AtEMP --- p.5 / Chapter 1.3 --- EMP70 Family Proteins --- p.5 / Chapter 1.3.1 --- General structure of EMP70 proteins --- p.5 / Chapter 1.3.2 --- EMP70 in other organisms --- p.8 / Chapter 1.3.3 --- EMP70 proteins in Arabidopsis --- p.9 / Chapter 1.4 --- Accession Numbers --- p.10 / Chapter 1.5 --- Research Objectives --- p.14 / Chapter Chapter 2 --- Generation and Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Selective AtEMP-GFP Fusions --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials and Methods --- p.17 / Chapter 2.2.1 --- RNA extraction and cDNA generation --- p.17 / Chapter 2.2.2 --- Construct making --- p.18 / Chapter 2.2.3 --- Bacterial strains --- p.21 / Chapter 2.2.4 --- Transformation of BY-2 cells --- p.21 / Chapter 2.2.5 --- Confocal fluorescence screening of tobacco BY-2 cells --- p.23 / Chapter 2.2.6 --- Drug treatments --- p.23 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Western blot analysis of tobacco BY-2 cell lines expressing AtEMP-GFP fusions --- p.25 / Chapter 2.3.2 --- Subcellular localization of AtEMP-GFP fusions to the PVC in transgenic BY-2 cells --- p.27 / Chapter 2.4 --- Summary --- p.30 / Chapter Chapter 3 --- Generation and Characterization of Antibodies Against Various AtEMPs --- p.31 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Materials and Methods --- p.33 / Chapter 3.2.1 --- Generation of antibodies --- p.33 / Chapter 3.2.2 --- Screening of antibodies --- p.36 / Chapter 3.2.2.1 --- SDS-PAGE and western blot analysis --- p.36 / Chapter 3.2.2.2 --- Confocal immunofluorescence studies --- p.38 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- AtEMP antibodies recognized EMP70 proteins in plant cells --- p.39 / Chapter 3.3.2 --- Organelles marked by anti-AtEMPs are closely associated with the Golgi apparatus --- p.40 / Chapter 3.4 --- Summary --- p.49 / Chapter Chapter 4 --- Subcellular Localization of GFP-tagged AtEMP Fusions via Transient Expression --- p.50 / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.2 --- Materials and Methods --- p.52 / Chapter 4.2.1 --- Making of transient expression constructs --- p.52 / Chapter 4.2.2 --- Transient expression --- p.57 / Chapter 4.3 --- Results --- p.59 / Chapter 4.3.1 --- PVC localization of AtEMP-GFP fusions --- p.59 / Chapter 4.3.2 --- Golgi localization of GFP-AtEMP and GFP-AtEMP-S fusions --- p.62 / Chapter 4.4 --- Summary --- p.66 / Chapter Chapter 5 --- Immunogold Electron Microscope Localization of AtEMPs --- p.67 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.2 --- Materials and Methods --- p.68 / Chapter 5.2.1 --- High-pressure freezing / freeze substitution --- p.68 / Chapter 5.2.2 --- Ultra-thin sectioning --- p.69 / Chapter 5.2.3 --- Immunogold labeling --- p.69 / Chapter 5.2.4 --- Post-staining and transmission election microscopy --- p.69 / Chapter 5.3 --- Results and Summary --- p.70 / Chapter Chapter 6 --- Discussion and Future Perspectives --- p.74 / Chapter 6.1 --- Hypothesis --- p.75 / Chapter 6.2 --- Subcellular localization of AtEMPs --- p.76 / Chapter 6.2.1 --- GFP-tagged AtEMP fusions --- p.76 / Chapter 6.2.2 --- Endogenous EMP70 proteins in BY-2 cells --- p.77 / Chapter 6.3 --- Targeting motifs in AtEMPs --- p.79 / Chapter 6.4 --- Conclusions --- p.81 / Chapter 6.5 --- Future perspectives --- p.82 / Chapter 6.5.1 --- Targeting motifs --- p.82 / Chapter 6.5.2 --- Functional studies --- p.82 / References --- p.83