No description available.

The HPr proteins were characterized as folding by a two-state folding mechanism. Here, we present a comparison of the equilibrium and kinetic folding for the HPr protein from Bacillus subtilis, E coli and a key variant from these proteins. For the wild-type protein we find that GHX is greater than GUDC, suggesting that the HPr does not fold by a simple two-state mechanism. This discrepancy is revealed by testing the two-state nature of the folding reaction of HPr with mutation. We show that removing a single charge side chain (Asp 69) converts the HPr protein back to a simple two-state mechanism. Ribonuclease Sa and two charge-reversal variants can be converted into amyloidin vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of ivwell-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.

The individual and combined effects of the endogeic and epigeic groups of earthworms on the growth of Chinese cabbage (Brassica rapa), and on the subsequent growth and development of the generalist sap-sucking herbivore Myzus persicae were determined in separate pot experiments. Many previous studies have investigated the relationship between soil biodiversity and aboveground plants, but few researches have considered the indirect interaction between soil biota and above-ground aphids. In this study the individual effects of Aporrectodea rosea and Allolobophora chlorotica and the combined effects of A. rosea and A. chlorotica, Aporrectodea caliginosa and Satchellius mammalis, A. chlorotica and S. mammalis, and A. rosea, A. caliginosa, Lumbricus rubellus and S. mammalis on plant morphology and physiology and aphid development (nymphs day-1, fecundity and number of adults) were investigated. Plant growth was affected by the presence of A. rosea which caused increases in plant biomass, height of plant, leaf surface area and specific leaf area (SLA). Mean number of leaves per plant was unaffected by worm density. In contrast, increasing density of A. chlorotica had no effect on any aspect of plant performance. The combined effect of A. rosea and A. chlorotica resulted in a similar increase in plant biomass to A. rosea alone. While the combined effects of A. caliginosa and Satchellius mammalis, A. chlorotica and S. mammalis and A. rosea, A. caliginosa, L. rubellus and S. mammalis caused increases in all plant parameters except leaf number. Additionally, the individual effect of A. rosea and the combined effects of A. caliginosa and S. mammalis, A. chlorotica & S. mammalis and A. rosea and A. caliginosa, L. rubellus and S. mammalis resulted in increased in leaf nitrogen concentration. Aphid development was also affected by the presence of earthworms. The nymphs day-1, fecundity and numbers of adults were significantly increased with increases in earthworm densities. The interaction between all groups of earthworms and their influence on aphid growth showed that the combined effect of two different groups was greater than the individual groups. Proteomic techniques were used to compare protein patterns in the plants. The combined effects of A. caliginosa and S. mammalis, A. chlorotica and S. mammalis, A. rosea, A. caliginosa, L. rubellus and S. mammalis earthworms on plant resulted in differences in number and kind of protein between plant treated with earthworms and the control, but no significant difference in proteins volume. Effects of earthworms on plant growth and aphid development are shown to be modified by increasing density and interactions between different species and functional groups of earthworms.

Fc-fusion proteins (e.g. EPO-Fc) are the most often created fusion proteins due to their beneficial biological and pharmacological properties. The economic success of Fc-fusion proteins and other biopharmaceuticals production however, greatly depends on a robust, low-cost and highly effective protein mammalian cell extraction system . Understanding of how cells respond to a protein production environment based on metabolic profiles provides new goals for bioengineering of cell lines for best performance in biomanufacturing. Furthermore, insights on how individual cell metabolism and therefore phenotype, respond to cell microenvironment allows the underlying biological mechanisms to be explored in greater detail. This study focused on the application of mass spectrometry (MS) technologies, combining the analysis of metabolic profiles of cells extracts by GC-MS and MALDI-MS and spatial visualisation and distribution of metabolites within cells producing the fusion protein by MALDI-MSI and SIMS imaging. The analysis of external and internal metabolome profiles of cells producing the protein showed an extended effect of EPO-Fc fusion protein production on cell metabolism. The findings indicate that changes observed in EPO-Fc producing cells are related to enhanced protein and lipid synthesis highlighting that these cells are in a state of increased metabolic activity with the protein exocytosis into growth medium. Moreover, the composition of lipid bilayer of induced cells seemed to be different to non-induced cells. These findings were confirmed with the analysis of EPO-Fc induced cells using MS metabolic imaging. Multivariate analysis highlighted a number of metabolites that were significantly influenced by the protein expression when compared to control cells. The major metabolic changes in induced cells were those related to lipid metabolism. The information about metabolic changes in tetracycline-induced cells obtained from the analysis of cell populations was further supported with the analysis based on single-cell studies. Single-cell based studies also proved that investigations of individual cells provide additional insights about changes in metabolism of induced cells that can be referred to a unique, single cell and its phenotype. The analysis of CHO cells revealed a high level of heterogeneity within a cell population. Different cell phenotype and hence, metabolite content allowed for correlation between cell locations and their metabolite characteristics, specific for each type of cells. This project has successfully shown combination of bio-analytical techniques to investigate external and internal metabolome changes related to a fusion protein production in mammalian cells. Additionally, the significance of single cell approaches in metabolomics has also been highlighted, providing insights into the sub-cellular distribution of metabolites in cells producing EPO-Fc and information on the level of heterogeneity within a cell population. A multidimensional approach for metabolic profiling and future technological improvements of single-cell platforms are required to provide improved data acquisition and data analysis in order to better understand unknown processes involved in cell metabolism.

One of the intriguing features of biological systems is the prevalence of highly selective and often very strong interactions among different cellular components. Such interactions play a variety of organizational, mechanical, and physiological roles at the cellular and organism levels. Antigen-antibody complexes are representative examples of highly selective and potent interactions involving proteins. The marked specificity of protein-antibody complexes have led to a wide range of applications in cellular and molecular biology related research. They have become an integral research tool in the present genomic and proteomic era. Unfortunately, the production of selective tools based on antigen-antibody interactions requires cumbersome protocols. The long term goal of this project explores the possibility of manipulating liposomes to serve as the chemical receptors ("artificial antibodies") against selected proteins. Cellular lipids (e.g., lipid rafts) are known to facilitate highly selective binding of proteins on cell membranes. The binding of proteins to cell membranes can be envisaged to be modulated via interactions between polar (charged) and non-polar head groups of lipids and the complementary amino acid residues of proteins. Their interaction is facilitated by a combination of van der Waals, electrostatic, hydrogen bonding and hydrophobic forces. A further interesting aspect of the above interaction is the "fluidity" of the membrane resident lipids, which can migrate from other regions to further enhance the complementary interactions of proteins on the initially "docked" membrane surface. With these features in mind, the end goal of this project is expected to deliver lipid-based chemical receptors "synthetically" designed against proteins to function as "artificial antibodies". Protein sensing will be accomplished with lipid receptors assembled in templated polymerized liposomes. The research presented here specifically focus on the analytical aspects of protein sensing via polymerized liposome vesicles. Lanthanide ions (Eu3+ and Tb3+) are incorporated into polymerized liposome with the expectation to "report" quantitative and qualitative information on the interacting protein. Our proposition is to extract quantitative and qualitative information from the luminescence intensity and the luminescence lifetime of the lanthanide ion, respectively. A thorough investigation is presented regarding the analytical potential of these two parameters for protein sensing. Two chemometic approaches - namely partial least squares (PLS-1) and artificial neural networks (ANN) - are compared towards quantitative and qualitative analysis of proteins in binary mixtures.

No description available.

This research developed a biosensor for kinase drug discovery applications. In particular it combined electronic techniques with optical techniques to understand the phosphorylation of proteins. There are two major electronic characteristics of phosphorylation that aid in its detection and subsequently biosensor development: first is the release of a proton upon phosphorylation of a protein (change in pH) and second is the addition of negative charge to the protein upon its phosphorylation. The work in this thesis reports an electrolyte–insulator–semiconductor sensing structures to detect the pH changes associated with phosphorylation and metal–insulator–semiconductor structures to detect the charge change upon phosphorylation of proteins. Major application of the developed devices would be to screen inhibitors of kinase that mediate phosphorylation of proteins. Inhibitors of kinase act as drugs to prevent or cure diseases due to the phosphorylation of proteins. With the advancements in VLSI and microfluidics technology this method can be extended into arrays for high throughput screening for discovering drugs.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: pp. 219-243.

No description available.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.