Life Sciences Commons^™

Mutational Optimization Of The Coelenterazine-Dependent Luciferase From Renilla, Jongchan Woo, Albrecht G. Von Arnim

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved k …

The Identification, Functional Characterization And Phylogeny Of The Nodulin-Like Anion Transporter (Nlat) Family In Plants, Eric Daniel Vincill

Doctoral Dissertations

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70). GmN70 is expressed predominantly in mature nitrogenfixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite >> chloride. These currents showed little sensitivity to pH or the nature …

Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations On Enzymes, Qin Xu

Doctoral Dissertations

The dynamic nature of proteins in solution is often an indispensable factor in biological function such as enzymatic catalysis. Complementary to the conventional structural analysis, computational simulations have the advantage to reflect the dynamic nature of proteins or enzymes. One of the computational simulation methods, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations, has been widely applied to the research in structural analysis, ligand-receptor binding and enzymatic catalysis.

In this dissertation, QM/MM MD simulations were applied to the studies on cytidine deaminase (CDA), yeast cytosine deaminase (yCD), and kumamolisin-As, as well as two protein lysine methyltransferases (PKMTs), DIM-5 and …

Contribution Of Water And Energetics Of Ligand Binding In The Catalytic Mechanism Of R67 Dihydrofolate Reductase, Shaileja Chopra

Doctoral Dissertations

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that …

Gene Alterations By Peroxisome Proliferator-Activated Receptor Gamma Agonists In Human Colorectal Cancer Cells, Maria Cekanova, X Li, J Yuan, K B. Kim, Seung J. Baek

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear transcription factor that controls the genes involved in metabolism and carcinogenesis. In the present study, we examined the alteration of gene expression in HCT-116 human colorectal cancer cells by PPARgamma agonists: MCC-555 (5 microM), rosiglitazone (5 microM), and 15-deoxy-Delta12,14-prostaglandin J2 (1 microM). The long-oligo microarray data revealed a list of target genes commonly induced (307 genes) and repressed (32 genes) by tested PPARgamma agonists. These genes were analyzed by Onto-Express software and KEGG pathway analysis and revealed that PPARgamma agonists are involved in cell proliferation, focal adhesion, and several signaling pathways. …

Mutational Optimization Of The Coelenterazine-Dependent Luciferase From Renilla, Jongchan Woo, Albrecht G. Von Arnim

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved k …