Cadmium (Cd2+) is a widely distributed industrial and environmental toxin , the effect of Cd2+, a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+-sensitive dye fura-2. Cd2+ increased [Ca2+]i in a concentrationdependent manner with an EC50 of 38

No description available.

Using protein design, a series of metal binding proteins have been designed, allowing the local factors that contribute to metal affinity and thermostability to be studied. Those proteins with the highest metal binding affinities had the lowest apo-form Tm and the largest ÄTm upon metal binding. In this thesis, major steps have been taken toward applying the engineered protein to MR imaging. The progress of magnetic resonance imaging is hindered by low specificity and rapid elimination of FDA-approved MRI contrast agents. The engineered protein contrast agent has been conjugated to a cancer-specific targeting peptide and encapsulated in albumin microspheres to provide tandem passive and active tumor targeting. Also, a simple, high-yield purification method has been developed.

Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins and peptide fragments. Using high resolution NMR, we have shown that the grafted EF-loop III of calmodulin in the host protein retains its native conformation with a strong loop and β-conformation preference. Grafted ligand residues in the engineered protein are directly involved in binding of Ca(II) and La(III). The NMR studies support our hypothesis that both ligand arrangement and dynamic properties play essential role in tuning Ca(II) binding affinities. Using pulse-field diffusion NMR and protein engineering, we further demonstrated that grafted EF- loop remains as a monomer. Although the EF-loop with flanking helices dimerizes in the presence of Ca(II). Additionally, removal of conserved hydrophobic residues at the flanking helices of the EF-hand motif leads to be monomer in the absence and presence of metal ions. Our results suggest that conserved hydrophobic residues are essential for the pair-paired interaction in the coupled EF-hand protein. We have shown that our developed grafting approach can be applied to probe intrinsic Ca(II) binding affinities of different Ca(II) binding sites.

This study was to investigate the reaction behavior of laboratory-prepared nanoscale adsorbents (Fe3O4 and H-[Fe3O4]MgO) and inorganic pollutants (NO3-, Cd2+and Cr6+) in simulated groundwater. First, Fe3O4 and the composites of nanoscale Fe3O4 and MgO were prepared using chemical co-precipitation method. Then they were characterized and verified by various apparatuses and methods including X-ray diffractometry, scanning electron microscopy, Zetasizer, and specific surface area measurements. Second, the nanoscale adsorbents were used to adsorb inorganic pollutants in simulated groundwater of different conditions. The relevant reaction behavior and mechanisms were also investigated. Results of this research showed that Fe3O4 and H-[Fe3O4]MgO had the greater adsorption amount when the initial concentration of inorganic pollutants was higher than lower. The adsorption rate of inorganic pollutants at 28¢J was greater than that of at 18¢J. The experimental results also showed that at a higher pH environment or the existence of humic acid in simulated groundwater would increase the removal efficiency of Cd2+, but decrease the removal efficiency of NO3- and Cr6+. Analysis of inorganic pollutants adsorption on nanoscale adsorbents in simulated groundwater indicated that a Langmuir-type of chemical adsorption and pseudo-second-order reaction kinetic equation would have better fit. In this study, it was also found that nanoscale adsorbents not only adsorbed inorganic pollutants but also reduced NO3- and Cr6+ to NO2-, NH4+, and Cr3+ at pH=3, respectively. Thus, the nanoscale adsorbents (Fe3O4 and H-[Fe3O4]MgO) prepared and were capable of reductively adsorbing inorganic pollutants (e.g., NO3- and Cr6+) for environmental remediation.

No description available.

Issue de la troncature de la dextrane-saccharase DSR-E, l’alpha-(1→2) transglucosidase recombinante GBD-CD2 catalyse à partir de saccharose le branchement de molécules acceptrices tels que les dextranes, les isomalto-oligosaccharides ou les gluco-oligosaccharides (GOS ; [6)-alpha-D-Glcp-(1→]n-alpha-D-Glcp-(1→4)-D-Glcp, avec 1<n<9). L’objet de cette étude a porté sur la compréhension des relations structure-activité de GBD-CD2 afin d’investiguer les facteurs structuraux responsables de la synthèse des liaisons osidiques de type alpha-(1→2). La troncature rationnelle du domaine de liaison au glucane (GBD) de l’enzyme GBD-CD2 (192 kDa) a abouti à l’isolement de trois formes tronquées actives, de masses moléculaires égales à 180, 147 et 123 kDa. Après purification de GBD-CD2 et de delta N123-GBD-CD2 (123 kDa), des études cinétiques ont permis de mettre en évidence que les enzymes présentent la même régiospécificité. L’activité d’hydrolyse du saccharose peut être modélisée par le modèle de Michaelis – Menten (kcat respectifs de 109 et 76 s-1). En présence de dextrane accepteur, ces enzymes sont activées. L’activité d’alpha-(1→2) glucosylation suit un modèle Ping Pong Bi Bi (kcat respectifs de 970 et 947 s-1). En modulant le ratio molaire entre le donneur d’unités glucosyle et l’accepteur de ces unités ([saccharose]/[dextrane]), il est possible de synthétiser des dextranes dont le pourcentage de liaisons alpha-(1→2) est contrôlé et varie de 10% à 40%. La caractérisation des produits de la réaction menée en présence de saccharose et de GOS a permis d’isoler et de caractériser pour la première fois des GOS arborant des unités glucosyle branchées en alpha-(1→2) sur les unités glucosyle adjacentes de la chaîne principale. Enfin, la résolution de la structure de delta N123-GBD-CD2 à 3,2 Å révèle que cette enzyme adopte le repliement original « en U » similaire à celui décrit pour GTF180-delta N. La comparaison des gorges catalytiques des deux dextrane-saccharases cristallisées apporte des éléments pouvant expliquer la régiospécificité singulière de delta N123-GBD-CD2, et ouvre la voie à des travaux de mutagenèse visant à investiguer le rôle de résidus potentiellement clés / GBD-CD2 is a recombinant alpha-(1→2) transglucosidase constructed by truncation of the DSR-E dextransucrase from Leuconostoc mesenteroides NRRL B-1299. From sucrose, GBD-CD2 catalyses the alpha-(1→2) branching reaction onto acceptor molecules such as dextrans, isomalto-oligosaccharides or gluco-oligosaccharides (GOS; [6)-alpha-D-Glcp-(1→]n-alpha-D-Glcp-(1→4)-D-Glcp, 1<n<9). This work has been focused on structure activity relationship studies. Rational truncations of the glucan binding domain (GBD) led to the expression in E. coli of three active enzymes, showing molecular masses of 180, 147 and 123 kDa. After purification of the recombinant GBD-CD2 and delta N123-GBD-CD2, we showed that both enzymes display the same regiospecificity. Steady-state kinetics revealed that the activity of sucrose hydrolysis displays a Michaelis Menten type of kinetics (kcat 109 s-1 and 76 s-1, respectively). In the presence of dextran acceptor, these enzymes are activated. The alpha-(1→2) transglucosidase activity from sucrose onto dextrans was modelled by a Ping Pong Bi Bi mechanism (kcat 970 s-1 and 947 s-1, respectively). When varying the molar ratio between the glucosyl donor and the acceptor ([sucrose]/[dextran]), the percentage of alpha-(1→2) linkages in dextrans can be controlled from 10% to 40%. Additionally, from reactions in the presence of GOS and sucrose, we isolated and characterized new alpha-(1→2) branched GOS with contiguous alpha-(1→2) branchings along linear GOS chains. Finally, the X-ray structure of delta N123-GBD-CD2 at 3.2 Å resolution revealed that this enzyme has a very original “U folding” similar to that described for GTF180-delta N. Study of the residues lining the catalytic gorges of the two crystallized enzymes revealed the structural determinants possibly involved in the singular regiospecificity of delta N123-GBD-CD2. Our work opens the way to mutagenesis work for discovering key structural determinants of delta N123-GBD-CD2

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 169-215.