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.

Session ID: TH-F3-4 / SCIS & ISIS 2010, Joint 5th International Conference on Soft Computing and Intelligent Systems and 11th International Symposium on Advanced Intelligent Systems. December 8-12, 2010, Okayama Convention Center, Okayama, Japan

Folding empowers the user to change the form and function of a sheet of paper through a sequence of manipulations. Unfolding the once folded artefact produces a diagram that describes its own making that can be replicated at different scales using a new material. Architecturally, folding can be employed a morphogenetic solution to design a system that can be fabricated from a sheet material, that like paper, can be folded into a inhabitable structure. The ease and cost efficiency of fabrication based on folding can be used to design a system that executed using low cost materials can be used as a shelter that accommodates programmatic and aesthetic evolution. Thus, the system lends itself to being a transitional shelter for communities that have been displaced due to a natural disaster or other form of crisis. Technological advances in design and structural analysis can give the designer the power to define the complex process folding parametrically allowing the input a real-time feedback based design based on an a folding inspired algorithm.

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.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.