Graduation date: 1997

In this study, sharp/gradual converging-diverging microchannels with contraction/ expansion ratio of 4:1/1:4 was designed to generate elongational flow with uniform velocity in the centerline. The £f-DNA stained with YOYO-1 was observed in the flow. MPIV was built to measure the velocity distribution and local strain rate was estimated by MPIV measurements. The deformation and conformation of individual DNA molecules in the flow was visualized with confocal laser scanning microscopy (CLSM). The goal of the present work was to develop a method for stretching DNA molecules, in order to perform analysis of coil-stretch transition of DNA. By measuring dynamic properties and relaxation time of DNA molecules stretched by pressure driven at various flow rate and viscosity, we have shown how one could investigate the influence of hydrodynamic interactions in the case of stretching of DNA molecules.

The conformational preferences of several oligosaccharides are investigated herein using a combination of NMR spectroscopy and molecular dynamics (MD) simulations, focusing on the torsion angles associated with the glycosidic linkages. Strategies for obtaining usable J-HMBC spectra for carbons with an adjacent 13C label are described. By employing a selective pulse or a constant time modification, spectra free from interferences are obtained for site-specifically 13C labeled oligosaccharides. Intermolecular hydrogen bonding in sucrose is investigated using MD simulations performed at different concentrations. One of the most frequent intermolecular hydrogen bonds in the simulations, O3f∙∙∙HO3g, was detected using the HSQC-TOCSY NMR experiment. Based on MD simulations and NMR spectroscopy, the conformational ensemble for a trisaccharide segment of the LeaLex hexasaccharide is proposed to feature conformational exchange between conformations with positive and negative values for the ψ3 torsion angle in the β-D-GlcpNAc-(1→3)-β-D-Galp linkage. Using MD simulations, the conformation of the N-acetyl group is shown to influence the glycosidic conformation at a nearby linkage in two oligosaccharides. Short (1→6)-linked oligosaccharides are shown to exhibit conformational exchange at the ω and ψ torsion angles. Notably, the former torsion angle populates states with ψ ≈ ±90°. Conformationally sensitive homo- and heteronuclear coupling constants are determined using various NMR experiments. The experimental data, including effective distances from NOESY obtained for two of the compounds, is used to improve the representation of the ω torsion angle in the CHARMM36 force field. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 5: Accepted. Paper 6: Manuscript.</p>

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Protein-protein and ligand-protein interactions as well as the effect of temperature and applied pressure on protein conformation were investigated by Fourier transform infrared (FTIR) spectroscopy with the use of Fourier self-deconvolution, Fourier derivative, and two-dimensional correlation techniques. The effect of the binding of biotin to avidin on the conformation of the protein was examined. At a biotin concentration of 0.05% (w/v), avidin was stabilized against thermal and pressure-induced denaturation. The IR spectra of avidin recorded as a function of increasing hydrostatic pressure in the presence and the absence of biotin indicated that, in the presence of biotin, avidin adopts a very compact structure in solution. The 2D correlation analysis of the infrared spectra of avidin recorded as a function of increasing temperature over the range of 25--95°C revealed that the protein unfolded via different pathways in the presence and the absence of biotin. Furthermore, the thermally induced conformational changes observed in the absence of biotin were irreversible upon cooling of the protein solution, owing to aggregation of the thermally unfolded protein, whereas in the presence of biotin the protein unfolded and refolded via the same pathway. Variable-temperature FTIR spectroscopy and 2D correlation analysis were also employed to examine correlations between absorptions in the amide I and amide III regions in the spectra of four proteins. The results suggested that the band at 1284 cm-1 in the amide III region may be tentatively assigned to the intermolecular beta-sheet structure formed upon aggregation of thermally unfolded protein. FTIR studies were also conducted to investigate the interaction between the catalytic and regulatory subunits of the enzyme ATCase. The secondary structure of the ATCase holoenzyme was stable up to 60°C. The isolated regulatory subunit denatured at approximately 47°C while the isolated catalytic subunit was thermall

Artemisia Sphaerocephala Krasch (ASK) polysaccharide, extracted from the outer layer of ASK seeds, it has attracted attention due to its reported bioactivities. In the present study, extraction and fractionation methods for ASKP were developed. Two different Mw fractions 60P (MW: 551 KDa) and 60S (MW: 39 KDa) were obtained using ammonium sulphate precipitation. The physicochemical properties, in terms of rheological properties, chemical compositions and surface activities, of both fractions were elucidated. Using high performance anion exchange chromatography (HPAEC), partial acid hydrolysis, methylation analysis, 1D & 2D NMR spectroscopy and MALDI-TOF-MS methods, the detailed structural features of 60P and 60S were obtained, which were confirmed to be arabino(glucurono)xylan and (galacto)glucomannan, respectively Using light scattering and HPSEC techniques, the conformational properties of 60P were investigated. 0.5 M NaOH solution exhibited better ability to eliminate aggregates of 60P compared to water, NaCl solution and urea solution. The molecule of 60P exhibited a random coil conformation and semi-flexible structure in 0.5 M NaOH solution. In addition, the dependence of conformation of 60P on the molecular weight was also revealed by HPSEC analysis. The computer modeling technique using RIS-Metropolis Monte Carlo (RMMC) method was also applied. The conformational properties of 60P with different T-GlcpA ratio and various distribution patterns (random, alternative and block) along the xylan chain were determined. Results indicated that: the more regular the side chain distribution, the more extended the structure. The conformational properties of 60S with different molecular weights and various ratios of O-acetyl group to mannose were also considered. Results indicated that the radius of gyration (Rg) demonstrated an increasing trend with Mw and the data was comparable with that obtained from HPSEC; the insertion of O-acetyl group increased the chain extension at low ratio while showing a decrease it at a high ratio. The present study provides fundamental information regarding the physicochemical properties, structural and conformational properties of polysaccharides from the seeds of Artemisia sphaerocephala Krasch (ASK). Such information is essential to the understanding of the structure-conformation- functionality relationships of ASKP, thus promotes their potential food and industry applications.

No description available.

Mimicking the structure and even the function of an ??-peptide with artificial chainmolecules such as ??-peptides, ??-peptides and other unnatural oligomers has shown early success.The structural similarities between natural peptides and oligoureas lead to the belief that C2-symmetric oligoureas could be a good candidate for peptidomimicry. Molecular modelingindicates that both homochiral (all monomers have the same absolute configuration) andalternate chiral (absolute configuration of the residues alternate) C2-symmetric oligoureas canform helix- and sheet-like structures in solution conditionally.Several C2-symmetric 1,2-diamines were chosen as the building blocks for the synthesisof chiral oligoureas, and all diamines except for one were prepared in lab. Homochiral andheterochiral oligoureas based on the same diamine or mixed diamines were synthesized in thesolution phase, growing a chain by adding one unit at a time to one terminus or two units at atime to both termini with 4-nitrophenoxycarbonyl (PNP)- activated and t-butoxycarbonyl (Boc)- protected diamines as the intermediates. All the chiral oligoureas were purified by eitherrecrystallization and /or column chromatography and/ or HPLC and characterized by NMR andMALDI-MS. For some oligoureas, crystal structures were obtained. Fragment condensation wasattempted to improve the efficiency of the synthesis, but this approach led to cyclized oligoureasinstead of the desired concatenated residues.Conformational studies of chiral oligoureas were done in both the solid state and thesolution state. The crystal structures of some homochiral oligoureas and some alternate chiraloligoureas indicate that both helix-like structures and extended structures exist for these C2-symmetric oligoureas. NMR and Circular Dichroism (CD) were used to study the conformationof oligoureas in solution, but the conformational study by NMR was not conclusive. CD studyshowed that these oligoureas have multiple conformations in solution and that some of theconformations are sensitive to solvents and temperature. Also, short homochiral and alternatechiral oligoureas based on trans-1,2-diaminocyclohexane (DACH) exhibit signs of cooperativebehavior in solution as gauged by a series of experiments.

Graduate / 0485

Graduate / 0485