To induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen¡¦s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies.

In this study, an electrokinetics-induced elongation flow was created inside a gradual converging-diverging microchannel with different temperature (25, 35, 45, 55¢XC). The conformation of DNA molecules, local strain rate, and the relaxation time play important roles in determining the extent of DNA stretching. By using £gPIV/£gLIF measurements, the velocity/temperature distributions in microchannels can be secured. The local strain rate was estimated by £gPIV measurements. We observe the hydrodynamic stretching DNA molecules in elongation flow by confocal laser scanning microscope (CLSM). Through CLSM images analysis, relaxation time of DNA molecules was estimated. Finally, dynamic properties and stretching ratio of DNA molecules stretched by EOF driven at various electric field and temperature ware measured. The thermal effect and the electric field on the conformation were also studied and discussed.

Ph.D.

No description available.

Acrylic free radical polymerization at high temperature proceeds via a complex set of mechanisms, with many rate coefficients poorly known and difficult to determine experimentally. This problem is compounded by the large number of monomers used in industry to produce coatings and other materials. Thus, there is a strong incentive to develop a methodology to estimate rate coefficients for these systems. This study explores the application of computational chemistry to estimate radical addition rate coefficients for the copolymerization of acrylates, methacrylates and styrene. The software package Gaussian is used to calculate heats of reaction (ΔHr) values for monomer additions to monomeric and dimeric radicals, using minimum energy structures identified and characterized for the reactants and products. The Evans-Polanyi relationship is applied to estimate reactivity ratios from the relative differences in ΔHr. The validity of this methodology is tested through a comparison of calculated monomer and radical reactivity ratios for acrylate, methacrylate, vinyl acetate, ethene and styrene systems to available experimental data for copolymerization systems. The methodology is found to work for some systems while there is computational breakdown in others due to steric crowding and/or breakdown of the Evans-Polanyi relationship. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2008-04-25 16:13:12.091 / NSERC

Although the native conformation of a protein is thermodynamically its most stable form, this stability is only marginal. As a consequence, globular proteins have a certain amount of flexibility in their backbones which allows for conformational changes in the course of their biological function. In the course of this thesis, we study protein models at the edge of stability in different contexts: (1) First, we use molecular dynamics to determine the force needed to rupture a chain molecule (an unfolded protein) being stretched at constant loading rate and temperature. When all energy bonds of the molecule are identical, we find that the force F depends on the pulling rate r and temperature T according to F ~ const -- T 1/3|ln(r/T)|1/3 When a single weak bond is introduced, this result is modified to F ~ const -- T2/3|ln(r/ T)|2/3 This scaling, which is model independent, can be used with force-spectroscopy experiment to quantitatively extract relevant microscopic parameters of biomolecules. (2) Second, we study the structural stability of models of proteins for which the selected folds are unusually stable to mutation, that is, designable. A two-dimensional hydrophobic-polar lattice model is used to determine designable folds and these folds were investigated under shear through Langevin dynamics. We find that the phase diagram of these proteins depends on their designability. In particular, highly designable folds are found to be weaker, i.e. easier to unfold, than low designable ones. This is argued to be related to protein flexibility. (3) Third, we study the mechanism of cold denaturation through constant-pressure simulations for a model of hydrophobic molecules in an explicit solvent. We find that the temperature dependence of the hydrophobic effect is the driving force for cold denaturation. The physical mechanism underlying this phenomenon is identified as the destabilization of hydrophobic contact in favor of solvent separated configurations, the same mechanism seen in pressure induced denaturation. A phenomenological explanation proposed for the mechanism is suggested as being responsible for cold denaturation in real proteins.

As a structural analogue of adenine (6-aminopurine), 2-aminopurine (2AP) is a powerful fluorescent probe, when substituted in DNA in place of the natural adenine. Time-resolved fluorescence measurements of 2AP-labeled oligonucleotides, together with steady-state spectroscopy give us an in-depth view of DNA-enzyme interactions, especially the conformational dynamics in solution phase. Herein, this technique has been extended to the study of the transient unzipping of DNA bases, to investigate the structure of three-way junction (3WJ), and the role of base unzipping in the mechanism of human flap endonuclease (FEN). Seven 2AP-labelled 3WJs were investigated, each containing only one 2AP base in place of adenine. In four of the 3WJs, 2AP was placed in the long duplex region of an arm; while in the other three 3WJs, 2AP was placed near or in the branch point. Comparative time-resolved fluorescence measurements on the 3WJs and corresponding ssDNA and dsDNA controls were made to study the base dynamics, in particular the possibility of unzipping in the vicinity of the branch point. In combination with single-molecule FRET measurements and molecular dynamics simulations, the local and global structure of a DNA 3WJ in solution could be unraveled. It was found to adopt a Y-shaped, pyramidal structure, in which the bases adjacent to the branch point are unzipped, despite the full Watson-Crick complementarity of the molecule. Human flap endonuclease (hFEN) is divalent metal ion-dependent phosphodiesterase. hFEN carries out structure-specific hydrolysis of 5’ bifurcated DNA endonucleolytically. Cleavage occurs at a position one nucleotide into the downstream duplex region. Previous structural, biochemical and modeling studies suggested a double-nucleotide unzipping mechanism at single/double strand junctions for scissile phosphate placement. To confirm this mechanism, 2AP time-resolved fluorescence spectroscopy was used to investigate nucleotide unzipping in hFEN substrates. 2AP was substituted at positions +1 and -1 (relative to the scissile phosphodiester) respectively, in double flap substrates. A series of hFEN mutants including Y40A, R100A, K93A, were used in this study. In the experiments, ssDNA, dsDNA substrates, DNA substrate-enzyme complexes were investigated in order to elucidate the enzyme-induced distortion of the substrate at the +1 and -1 positions. TseI is a thermophilic type II restriction enzyme which has ideal activity at an elevated temperature. It is able to recognise and cut the 5 bp palindromic sequence of 5’-GCWGC-3’ (W=A or T). A range of biophysical methods have been applied to investigate this enzyme, including size-exclusion chromatography; fluorescence anisotropy (Kd value determination); denaturing HPLC for DNA cleavage analysis on matched and mismatched substrates; fluorescence-based activity assay (KM, Vmax, kcat, specificity constant values determination); steady-state fluorescence measurements (DNA-enzyme interaction study). The DNA cleavage characteristics of TseI were fully studied and it was found that it cuts A:A and T:T mismatches in CAG and CTG repeats. This potentially makes it a useful tool for exploring unusual DNA structures containing super-long CAG and CTG repeats which are involved in the aetiology of some neurodegenerative diseases, such as Huntington’s disease (HD). EcoP15I is a type III restriction-modification enzyme whose recognition sequence is 5-CAGCAG-3’. Methyltransferase EcoP15I (M.EcoP15I) adds a methyl group to the second adenine, in the presence of cofactor S-adenosyl methionine (SAM). SDS-PAGE, densitometry and size-exclusion HPLC were applied to confirm that EcoP15I adopts a Res1Mod2 stoichiometry in solution. The large structural distortion of its substrate (base flipping) by M.EcoP15I was investigated by both steady-state and time-resolved fluorescence. Also, nine 120 mer DNA duplexes, each containing two reversely oriented recognition sites were used to study matched and mismatched sequence cleavage by R.EcoP15 and a cleavage pattern was revealed.

A synthetic analog of a spider silk protein (M4) was studied. Langmuir films were made and an inflexion in the isotherm indicated conformational changes upon compression. Deposition onto solid substrates was most successful using a hydrophobic substrate and the Langmuir-Schaeffer method. AFM was used to image the surface, which was mesh like and did not show any indication of order. / Gold nanoparticles were produced in the presence of the protein and protein solutions were added to read made nanoparticles for the purpose of displacing the weak ligands present. CD measurements were performed on the protein solutions to study its conformation. Nanoparticle size information was obtained from TEM images. DLS was used to determine if the protein was affected by the addition of the gold nanoparticles. Precipitation of the protein was shown not to affect the nanoparticles.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.