Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 193-199). Available also in a digital version from Dissertation Abstracts.

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The twin-arginine translocase allows the translocation of folded protein substrates across the cytoplasmic membrane of bacteria and archaea or the thylakoid membrane of plants. In Escherichia coli, its protein components TatA, TatB and TatC assemble dynamically upon interaction with protein substrates. Prior to export, the machinery performs a quality control check so that only correctly folded proteins are translocated. The first objective of this work was to derive and apply new methodologies based on the inherent qualities of the pathway. We developed a new bacterial two-hybrid system that capitalizes on the folding quality control mechanism of the Tat pathway. One protein (prey) is fused to Tat-specific signal peptide. A second (bait) protein is produced as a fusion to a reporter that produces a "signal" (growth or enzymatic activity) only when the bait-reporter fusion binds to the prey and the resulting complex is exported into the periplasm via the Tat pathway. As a second biotechnological application of the Tat pathway, we developed a phage display system that allows the protein of interest to fold within the cytoplasm prior export and display onto phage particles. This is in contrast to the conventional phage display system, in which displayed protein folds in the periplasm. We took advantage of this new system to screen a library of 2 x 10⁶ of fluorescent GFP variants containing a hexameric peptide insertion for ligand binding. Despite the diversity of the hexamer, we were not able to isolate single GFP variants that would bind with specificity to various ligands. This highlights the difficulty in engineering GFP variants that can bind to other proteins while retaining the ability to fluoresce. The second aspect of this research was to examine mechanistic aspects of the Tat pathway. TatB and TatC are responsible for the recognition of Tat signal peptides. Here, we established the importance of TatC as the crucial component of the Tat pathway for the interaction with the hallmark twin-arginine motif within Tat signal peptide. Substitution of the RR dipeptide with a KK sequence completely abolishes export. In a genetic screen using a ssTorA(KK)-GFP-SsrA as a reporter. We identified several amino acid substitutions within TatC that allowed the alteration of the substrate specificity of the pathway as indicated by the impairment of indigenous Tat substrates. Finally, we analyzed the conformational dynamics of TatA using GFP fusions and by incorporation of the chemically reactive, non-canonical amino acid azidohomoalanine.

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study, Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted with 3-fluorophenylalanine. 19F NMR studies then focused on properties of the hydrophobic core associated with the folding process at temperatures near the regime where the protein is completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19F NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with increasing temperature, until the point at which the near-native intermediate state is attained. At this point, water is observed to enter the hydrophobic core and destabilize the protein. Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with temperature until the near-native intermediate is reached, whereupon oxygen solubility decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with temperature, until a dry near-native state is established, whereupon water cooperatively enters and destabilizes the hydrophobic core. 19F CPMG experiments provide a measure of the interconversion between the folded state and the dry near-native intermediate; at higher temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered, folding rates increase, presumably because the effect of off-pathway misfolding events on the exchange process is diminished.

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study, Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted with 3-fluorophenylalanine. 19F NMR studies then focused on properties of the hydrophobic core associated with the folding process at temperatures near the regime where the protein is completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19F NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with increasing temperature, until the point at which the near-native intermediate state is attained. At this point, water is observed to enter the hydrophobic core and destabilize the protein. Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with temperature until the near-native intermediate is reached, whereupon oxygen solubility decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with temperature, until a dry near-native state is established, whereupon water cooperatively enters and destabilizes the hydrophobic core. 19F CPMG experiments provide a measure of the interconversion between the folded state and the dry near-native intermediate; at higher temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered, folding rates increase, presumably because the effect of off-pathway misfolding events on the exchange process is diminished.

Protein folding pathways can be extraordinarily complex. In this study, circular dichroism (CD) and 19F NMR are used to investigate the folding network of calmodulin, a calcium-binding protein, which is biosynthetically enriched with 3-fluorophenylalanine. In calmodulin’s calcium-loaded state, CD experiments identify the existence of a folding intermediate along a heat-denaturation pathway. In comparison to the native state, 19F NMR solvent isotope shifts reveal decreased accessibility of water to hydrophobic core, whereas O2 paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N-1H and methyl 13C-1H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest that this near-native intermediate state is transiently adopted below the temperature associated with its onset. The folding network also involves an unproductive off-pathway intermediate. In contrast, calmodulin’s calcium-free state exhibits a simpler folding process which lacks discernible intermediates.

As nascent secretory and membrane proteins are inserted into the endoplasmic reticulum (ER), they are maintained in folding and/or assembly competent states by molecular chaperones including the Hsp70 and Hsp90 homologues, BiP and GRP94, and the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). Folding is catalyzed by protein disulfide isomerase (PDI), its CNX (and CRT) associated homologue, ERp57, and protein prolyl isomerase (PPI). Moreover, N-linked glycoproteins benefit from a lectin-based "quality control apparatus" that ensures their correct folding or oligomeric assembly. Binding to these lectins occurs through oligosaccharide trimming from Glc 3Man9GlcNAc2 to the monoglucosylated form (Glc 1Man9GlcNAc2). Release and subsequent rebinding occurs though the hydrolysis and reglucosylation of the innermost glucose by glucosidase II and UDP-glucose glycoprotein:glucosyltransferase, respectively. This cyclical process, termed the "Calnexin Cycle", continues until their correct conformation is achieved. / The cloning and characterization of human glucosidase II is reported here. cDNAs for two splice variants of the catalytic alpha subunit and the beta subunit were isolated. Expression of the beta subunit was shown to be required for enzymatic activity, solubility and/or stability, and ER retention of the enzyme. Detailed kinetic analysis on recombinant alpha1/beta and alpha2/beta isoforms, using p-nitrophenyl alpha-D-glucopyranoside as a substrate, reveals that both exhibit kinetic profiles of a two binding site model, and share properties of catalysis and inhibition on this substrate. Moreover, similar rates of hydrolysis of the oligosaccharide substrates rules out the possibility that the two binding site kinetic model, first proposed by Alonso et al. (1999, Biochem J. 278:721--7), is the result of co-purified isoforms of glucosidase II that have different substrate specificities. / Also, an ER protein two-hybrid system, based on Ire1p and the unfolded protein response (UPR) pathway in Saccharomyces cerevisiae, was developed to examine and map the interactions between CNX/CRT and ERp57. Ire1p fusions with CNX and CRT were shown to interact specifically with ERp57, and as expected, PDI did not. Through deletion analysis, new roles were assigned to the proline-rich loop domains of CNX and CRT, and the non-catalytic B thioredoxin domain of ERp57 in mediating their heterodimerization.

Protein folding pathways can be extraordinarily complex. In this study, circular dichroism (CD) and 19F NMR are used to investigate the folding network of calmodulin, a calcium-binding protein, which is biosynthetically enriched with 3-fluorophenylalanine. In calmodulin’s calcium-loaded state, CD experiments identify the existence of a folding intermediate along a heat-denaturation pathway. In comparison to the native state, 19F NMR solvent isotope shifts reveal decreased accessibility of water to hydrophobic core, whereas O2 paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N-1H and methyl 13C-1H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest that this near-native intermediate state is transiently adopted below the temperature associated with its onset. The folding network also involves an unproductive off-pathway intermediate. In contrast, calmodulin’s calcium-free state exhibits a simpler folding process which lacks discernible intermediates.

Thesis (Ph.D.)--Tufts University, 2001. / Adviser: David L. Weaver. Submitted to the Dept. of Physics. Includes bibliographical references (leaves 108-113). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007. / "A dissertation in cell biology and biophysics and molecular biology and biochemistry." Advisor: Antony A. Cooper. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 7, 2008 Includes bibliographical references (leaves 100-134). Online version of the print edition.