Browsing by Subject "Protein binding"
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Item Characterization and evolution of peridinin-chlorophyll a binding protein gene families in symbiotic dinoflagellates(2002) Reichman, Jay Randall; Hillis, David M.; Vize, Peter D.This dissertation consists of three integrated chapters. Chapter One presents the first genomic characterization of the PCP gene family from a symbiotic dinoflagellate, Symbiodinium sp. from Hippopus hippopus (Symbiodinium 203). Symbiodinium 203 has long PCP genes organized like those of Amphidinium carterae and Lingulodinium polyedra, but with a putative promoter that is different from L. polyedra. There are at least 14 distinct coding regions out of 36 ± 12 PCP genes in this family. Diversity of Symbiodinium 203's PCP gene family appears to be consequence of low levels of concerted evolution and acts as a primary source of variability in PCP isoforms. Predicted amino acid substitutions in Symbiodinium 203's PCP apoproteins result in shifts of isoelectric points, and protein modeling suggests that polymorphic sites may influence light harvesting of holoproteins. In Chapter Two, the first PCP coding sequences from S. pilosum, Symbiodinium sp. from Dichocoenia stokesii, S. pulchrorum and S. kawagutii were presented. Diverse PCP gene families occur in all major clades of Symbiodinium and in both size classes of the gene. As with Symbiodinium 203 in Chapter One, these PCP gene families do not appear to have been homogenized through mechanisms leading to concerted evolution. The predicted PCP apoproteins from S. pilosum and S. kawagutii have calculated isoelectric focusing points that generally match values previously measured for these species, which supports the hypothesis that genetic polymorphism is the primary source generating differences in PCP isoforms. Protein modeling produced a putative tertiary structure for S. pilosum apoproteins and was used to identify polymorphic sites in S. pilosum and S. kawagutii PCPs that could affect spectral tuning of peridinins. And Chapter Three contains the first phylogenetic analyses of the evolution of dinoflagellate PCP gene families. The objective of this section is to estimate the selective pressure at the codon level within PCP genes. PCP polymorphism is ancient, however, the polymorphism is not maintained by positive selection. Codon sites within PCP genes are evolving under purifying selection and are subjected to net reduced levels of concerted evolution. Isoform diversity is probably selected for within a functional range.Item 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 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 Purification and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae(1995-12) Maeng, Cheol-young; Not availableItem 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 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.