Browsing by Subject "Protein binding"
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Item Analysis of DNA-Binding Nonhistone Chromosomal Proteins(Texas Tech University, 1978-12) Lee, Shuey-ChyongNot Available.Item Applications of affinity chromatography to isolation of sequence specific nuclear proteins(Texas Tech University, 1981-05) Sherrod, Peter D.Even before Watson and Crick proposed their brilliant model for the structure of DNA, Jacob and Monod were busy piecing together the information that would lead to the first fully defined mechanism for genetic regulation in the bacteria, Escherichia coli. Since then, other mechanisms have become fell defined in prokaryotes, however eukaryotic mechanisms of control have been difficult to establish. This is primarily due to the fact that the genome of euiiaryotic cells is much more complex than in bacteria. Just the immensity of size defies the precise and controlled study that has yielded so much information in prokaryotic systems. Nevertheless, it is not inconceivable that similar mechanisms of regulation exist in the eukaryotic genome.Item Cation/amino acid symports in the photosynthetic bacterium Chromatium vinosum(Texas Tech University, 1984-05) Cobb, K. AndreaNot availableItem Characterization and evolution of peridinin-chlorophyll a binding protein gene families in symbiotic dinoflagellates(2002) Reichman, Jay Randall; Hillis, David M.; Vize, Peter D.Item Development and application of small molecule chaperones for protein renaturation(Texas Tech University, 2003-12) Liu, Xiangyi; Flowers II, Robert A.; Birney, David M.; Pare, PaulProtein aggregation presents a major problem in protein renaturation. This is due to competing intramolecular and intermolecular processes in unfolded protein molecules. Current investigations in our lab have focused on examining additives that can aid denatured polypeptide chains to refold into their native conformations and stabilize the structure of resulting proteins. Our approach to this problem is the development of a series of fluorous and non-fluorous salt additives that are capable of stabilizing protein structure against irreversible thermal denaturation, allowing protein molecules to refold into their native conformations, and recovering protein activities after chemical denaturation. Our initial studies employed Hen Egg White Lysozyme (HEWL) and Carbonic Anhydrase B (CAB) as model proteins to explore the effects of these salts on protein stabilities and renaturation. We also studied inclusion bodies of MMP 13 provided by Pfizer, Inc, and examined the utility of our protocol in a more practical application. The behavior of fluorous and non-fluorous salts prepared in our lab was investigated in the case of both thermal and chemical denaturation of HEWL and CAB. Differential Scanning Calorimetry (DSC) was employed to monitor the thermal process of protein solutions treated with salt additives. While many of those salts are able to prevent aggregation of HEWL during heating at a high temperature, fluorous salts were found to have an unusual stabilizing effect on protein structure. Moreover, compared with non-fluorous salts, fluorous salts could generate higher recovered enzymatic activities from chemically reduced-denatured HEWL at a useful concentration. The structure of recovered proteins was investigated further using Circular Dichroism (CD) spectroscopy. Although difficulties were encountered in attempting to prevent the irreversible thermal denaturation of CAB, fluorous salts have significantly enhanced the recovery of chemically denatured CAB molecules at a relatively high concentration. In our attempt to renature MMP 13 inclusion bodies, we obtained the results that further confirm the critical importance of the fluorous character in salt additives, hi addition, all the refolded-active proteins were separated from the salts by a simple dialysis protocol. Our approach to protein renaturation has a number of advantages. Furthermore, the observation of the important role of fluorous portion in stabilizing and recovering native protein structure provides the potential to develop a series of highly efficient additives for protein renaturation.Item DNA-binding proteins in Escherichia coli(Texas Tech University, 1977-05) Eaton, Leslie C.Not availableItem Exploiting aromatic donor-acceptor recognition in the folding and binding of naphthyl oligomers(2004) Gabriel, Gregory John; Iverson, Brent L.Biomolecules, for example, DNA and enzymes, perform nearly all the chemical processes essential for life. Their functions are dependent though on their ability to fold and bind into precise three-dimensional conformations and assemblies. A variety of oligomers that adopt compact conformations in solution, termed foldamers, have been synthesized to elucidate strategies to control folding and binding akin to biomolecules. The Iverson group has been developing a class of foldamers, called aedamers, which employ the aromatic-aromatic complexation between electronrich 1,8-dialkoxy-naphthalene (Dan) and electron-deficient 1,4,5,8-naphthalenevii tetracarboxylic diimide (Ndi) “building blocks”. It is expected that further work with these naphthyl oligomers will help establish aromatic interactions as a reliable tool for the construction of water-stable assemblies with tunable and predictable properties not found in nature. Overall, this dissertation describes the group’s first attempts to test the structural “designability” of naphthyl oligomers of previously unexplored sequences. Bottomline is that these studies have utilized the Dan:Ndi interaction to dictate intra- and inter- molecular associations to afford distinct folding topologies and achieve selective binding, respectively. Chapter 2 reports the observation that a previously studied amphiphilic aedamer happens to be an effective refolding inhibitor of RNase thus introducing the prospect of aedamer-protein interactions, a long-standing aim for these molecules. Chapter 3 presents the “shuffling” of the aedamer sequence (DanNdi)n to afford naphthyl oligomers, of the form Dann+1Ndin, that adopt turn structures. The results here demonstrate the ability of foldamers to access various secondary structures through changes to their primary sequence analogous to proteins. Chapter 4 details the first hetero-duplex system to operate via aromatic interactions in aqueous solutions. Dann and Ndin complementary strands exhibit high binding affinities and chain discrimination. The ability of the Dan:Ndi association to direct binding is expected to be extensively used by the laboratory to create discrete assemblies. As a whole, these projects probe the folding and binding of naphthyl oligomers in a variety of situations to demonstrate the wide reach of directed aromatic interactions to create various architectures. With this level of control established, surface patterning for microarrays, functional artificial proteins, biomolecule-aedamer ensembles, and other application-driven pursuits using naphthyl oligomers are possible in the near future.Item Further purification of DNA-binding nonhistone chromosomal proteins(Texas Tech University, 1977-08) Tsai, SueshunNot availableItem Identification of cubilin (p400) as galectin-3 binding protein from the murine utero-placental complex(Texas Tech University, 2000-12) Crider-Pirkle, Sunday Suzanne; Faust, Charles; Weitlauf, Harry M.; Hardy, Daniel; Lee, Vaughan H.; Webster, Daniel R.; Whelly, Sandra M.Galectin-3 is a soluble p-galactoside binding lectin that is present in several cell types within the uteroplacental complex (UPC) of mice. Affinity chromatography with immobilized galectin-3 was used to isolate potential binding partners for the lectin from homogenates of UPC. At least one glycoprotein (Mr 400,000; p400) was isolated that bound galectin-3 in a carbohydrate-dependent manner. Exposure of p400 to glycosidases decreased its apparent size by 10%. Differential migration of p400 in nonreducing and reducing conditions demonstrated that the protein contains intramolecular disulfide bonds. Amino acid sequencing revealed similarity to cubilin, a 400 kDa endocytic receptor. Collectively, the molecular size of p400, its degree of glycosylation, the presence of intramolecular disulfide bonds, and amino acid sequence similarity strongly suggest that p400 is the murine ortholog of cubilin. Immunohistochemistry revealed that cubilin (p400) was present in the yolk sac epithelium from day 8 to term. It was also localized in the perforin-positive granules of uNK cells in metrial gland and decidua basalis. Although cubilin is best known as the receptor for intrinsic factor-vitamin B12 in the ileum, it may also act as an endocytic receptor in the kidney and yolk sac where it presumably mediates transcytosis of multiple ligands. The localization of cubilin to uNK cells is the first demonstration of the protein in an immune or non-epithelial cell type. However, the questions of whether cubilin actually interacts with galectin-3 in vivo, and what role cubilin plays, cannot be answered from our results. Because both galectin-3 and cubilin are present in uNK cells, one intriguing hypothesis is that they interact to modulate the immune function of uNK cells, and thus, that they are a part of a mechanism for protecting the fetus from immune rejection.Item Mechanism and regulation of the protein kinase ERK2(2006) Callaway, Kari-Kristin Anderson; Dalby, Kevin N.The extracellular signal-regulated kinase 2 (ERK2) cascade plays important roles in a variety of cellular events such as proliferation, differentiation, and apoptosis. Involvement in such diverse cellular processes demands that this signal transduction pathway be strictly controlled. The fact that perturbations in the ERK2 signaling pathway are associated with a variety of diseases only further emphasizes the importance of maintaining the fidelity of the signaling cascade. In order to understand the mechanism of signal transduction fidelity, the kinetic mechanism and protein-protein interactions of ERK2 have been examined. In Chapter 1, a fluorescence anisotropy assay was developed to monitor the protein-protein interactions that occur between ERK2 and potential substrates. Results from these studies demonstrate that the phosphorylation status of ERK2 can alter protein-protein interactions. In Chapter 2, stopped-flow fluorescence spectroscopy studies demonstrate that the binding of EtsΔ138 to ERK2 follows a two-step mechanism in which binding occurs first followed by a conformational change. The results also suggest that substrate binding does not involve the active site, but instead occurs through the interaction of ERK2 docking-recruiting sites with EtsΔ138 docking motifs. In Chapter 3, transient kinetic methods were used to identify the second rate-limiting step in the ERK2 reaction pathway. The experiments provided evidence that an ADP dissociation step partially limiting enzymatic turnover. In Chapter 4, the magnesium effects on the catalytic mechanism of ERK2 were investigated. According to the study, ERK2 utilizes a second Mg2+ ion to facilitate ternary complex formation and catalysis, without inhibiting MgADP release, which is partially rate-limiting. The molecular basis for magnesium activation was found to stem from the interaction of the second Mg2+ ion by Asn-152. In Chapter 5, the interaction between ERK2 and PEA-15 was analyzed and PEA-15 was found to act as an inhibitor of ERK2- D-site interactions by binding to ERK2 via a proposed D-site motif. Further examination revealed that the neither the activation state of ERK2 nor the phosphorylation of PEA-15 has an affect on the affinity of these two proteins for one another.Item Molecular Topography and Binding Properties of Phytochrome and Other Related Tetrapyrrolic Proteins(Texas Tech University, 1987-05) Singh, Bal RamNot Available.Item Molecular Weight of Condensed Tannins from Warm-season Perennial Legumes and Its Effect on Condensed Tannin Biological Activity(2013-05-21) Naumann, Harley DeanCondensed tannins (CT) are polyphenolic compounds that have demonstrated biological activities in ruminants including suppression of enteric methane (CH4) production, protein binding and suppression of gastrointestinal nematode (GIN) infections. Some forage CT have been reported to be biologically active, whereas others have demonstrated no biological activity at all. While the chemical structure of CT has been postulated to be a key contributing factor affecting biological activity, the specific factors that determine whether or not CT from a specific forage have bioactive properties remain unknown. Results from previous studies have shown that as molecular weight of CT increases, CT biological activity also increases. Others have reported no effect of CT molecular weight on biological activity. The relationship between molecular weight of CT and CT biological activity remains inconclusive. The effect of molecular weight of CT from a variety of warm-season perennial legumes commonly consumed by ruminants on biological activity has not been adequately explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to suppression of enteric CH4 production, protein-binding ability (PB) and anthelmintic activity, and to compare the biological activity of CT from native warm-season perennial legumes to that of the introduced species Lespedeza cuneata, a plant that has gained attention in recent years due its anthelmintic properties. All or a combination of the following warm-season perennial legume species were evaluated for in vitro gas production, protein-precipitable phenolics (PPP) and PB, and percent larval migration inhibition (LMI). Eight North American native warm-season perennial legumes: Leucaena retusa Benth. (littleleaf leadtree), Desmanthus illinoensis (Michx.) MacMill. Ex B.L. Rob. & Fernald (Illinois bundleflower), Lespedeza stuevei Nutt. (tall lespedeza), Mimosa strigillosa Torr. & A. Gray (powderpuff), Neptunia lutea (Leavenworth) Benth. (yellow puff), two ecotypes of Acacia angustissima var. hirta (Nutt.) B.L. Rob (prairie acacia), Desmodium paniculatum (L.) DC. var. paniculatum (panicledleaf ticktrefoil), and two introduced legumes: Arachis glabrata Benth. (rhizoma peanut) and Lespedeza cuneata (Dum. Cours.) G. Don (sericea lespedeza) were included. In vitro CH4 production regressed on CT MW resulted in a R2 of 0.0009 (P = 0.80). There was no correlation between PPP or PB and MW of CT (R^2 0.11; P = 0.17 and R^2 0.02; P = 0.54, respectively). There was a weak correlation between CT MW and percent LMI (R^2 0.34; P = 0.05). The results of our study strongly suggested that CT MW does not explain the biological activities of enteric methane suppression or protein-binding ability. Condensed tannin MW may be involved in anthelmintic activity of CT from the forage legumes surveyed. North American native legumes containing biologically active CT, as compared to introduced species, were identified as having promise for use in ruminant diets.Item Regulation of effector caspases by inhibitor of apoptosis (IAP) proteins(2008-08) Choi, Young Eun; Bratton, Shawn B.Apoptosis is a biologically essential phenomenon executed in large part by caspases. Members of the caspase family are activated at different points during apoptosis to proteolyze specific substrates. Given that both excessive and insufficient apoptosis is related to the pathogenesis of various diseases, proper regulation of caspases and apoptosis is necessary for the health of living organisms. Inhibitor of apoptosis (IAP) proteins are endogenous inhibitors of caspases, and since XIAP, the prototypical IAP, binds to and inhibits caspases, all IAPs have been speculated to engage in similar inhibition mechanisms. However, in this dissertation, I demonstrate that cIAP1 binds to the effector caspases-3 and -7, through distinct mechanisms. cIAP1 readily binds to and ubiquitinates, but dos not directly inhibit the activity of fully mature caspase-7. By contrast, cIAP1 does not bind to caspase-3. cIAP1 binding to caspase-7 is mediated primarily by the N-terminus of the large subunit of caspase-7. An AKPD motif located on the N-terminus of caspase-7 is involved in the proteasome-mediated degradation of caspase-7 in cells, thereby decreasing the sensitivity of these cells to apoptosis. Thus, I demonstrate for the first time that cIAP1 is capable of inhibiting caspase-dependent apoptosis through indirect regulation of caspase activity.Item A structure/function analysis of macromolecular recognition by the protein kinase ERK2(2004) Rainey, Mark Allan; Dalby, Kevin N.Mitogen-activate protein kinases (MAPKs) phosphorylate protein substrates in the presence of magnesium and adenosine triphosphate in response to extracellular environmental signals to carry out signal-dependent intracellular responses. Extracellular signal-regulated protein kinase 2 (ERK2), a member of the MAPK family, mediates cellular growth, differentiation, and proliferation in response to growth factors. Understanding the mechanism by which MAPKs specifically recognize their protein substrates to carry out phosphoryl-transfer on specific residues within these macromolecules is critical for understanding the mechanism of signal transduction fidelity. Phage display was carried out against the active form of ERK2 to find novel ERK2-binding peptides. One peptide, KKKIRCIRGWTKDIRTLADSCQY, inhibited ERK2 phosphorylation of the protein substrate Ets∆138, exhibiting competitive and mixed inhibition towards Ets∆138 (Ki = 20.7 ± 5.5 µM) and MgATP2-, respectively. Steady-state kinetics combined with a novel fluorescence anisotropy binding assay were used to quantitatively elucidate the roles of several proposed ERK2 exosites in forming a macromolecular docking complex with Ets∆138 required for efficient phosphorylation. An ERK2–Ets∆138 docking complex (Kd of 6.6 ± 1.2 µM) was shown to form independent of the substrate phospho-acceptor. Docking motif peptides proposed to bind ERK2 exosites could dissociate the ERK2–Ets∆138 docking complex, however, dissociation did not occur using a peptide containing an ERK2 phospho-acceptor indicating the lack of active site interactions in the docking complex. Mutation of ERK2 residues Lys-229 and His-230 to p38 MAPKα-like residues, an enzyme that does not efficiently phosphorylate Ets∆138, led to a 20-fold decrease in the specificity constant (kcat/Km) of Ets∆138 phosphorylation largely due to its inability to bind Ets∆138. This structure/function analysis offers a quantitative approach towards understanding the molecular determinants of protein substrate recognition by a protein kinase prior to phosphorylation.Item The RING finger binding protein is a nuclear membrane protein that interacts with RUSH transcription factors(Texas Tech University, 2001-05) Mansharamani, Malini; Chilton, Beverly S.; Lee, Vaughan H.; Pfarr, Curt M.; Pressley, Thomas A.; Webster, Daniel R.; Whelley, Sandra M.Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH lα, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612-804 of the RFBP protein. Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.Item Towards peptide-binding peptides(2001-08) Zhang, Zhiwen; Anslyn, Eric V., 1960-; Kodadek, Thomas J.Peptide-binding peptides can be a very powerful research tool. A novel methodology, based on the mechanism of bacteriophage l switch in E. coli and combinatorial screening, has been developed to isolate peptides that bind another target peptide in vivo. Two pairs of interacting peptides have been isolated and characterized. One of the potential applications of such peptide-binding peptides is to be utilized as protein purification tags. Another novel aspect of this research is that a candidate peptide is able to inhibit an enzyme-catalyzed protein hydrolysis by binding specifically to a peptide sequence on the substrate which is recognized by the protease. In other words, a novel concept of substrate-directed enzyme inhibitors has been developed.