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- All Subjects: Biochemistry
- Creators: Gould, Ian
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
A potential new class of cancer chemotherapeutic agents has been synthesized by varying the 2 position of a benzimidazole based extended amidine. Compounds 6-amino-2-chloromethyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1A) and 6-amino-2-hydroxypropyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1B) were assayed at the National Cancer Institute's (NCI) Developmental Therapeutic Program (DTP) and found to be cytotoxic at sub-micromolar concentrations, and have shown between a 100 and a 1000-fold increase in specificity towards lung, colon, CNS, and melanoma cell lines. These ATP mimics have been found to correlate with sequestosome 1 (SQSTM1), a protein implicated in drug resistance and cell survival in various cancer cell lines. Using the DTP COMPARE algorithm, compounds 1A and 1B were shown to correlate to each other at 77%, but failed to correlate with other benzimidazole based extended amidines previously synthesized in this laboratory suggesting they operate through a different biological mechanism.
ContributorsDarzi, Evan (Author) / Skibo, Edward (Thesis advisor) / Gould, Ian (Committee member) / Francisco, Wilson (Committee member) / Arizona State University (Publisher)
Created2011
Description
The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.
ContributorsBozeman, Trevor C (Author) / Hecht, Sidney M. (Thesis advisor) / Chaput, John (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is to make a variety of mutant CV-Ns to specifically bind other glycan targets. Such a set of lectins may be used as screening reagents to identify biomarkers and other glycan motifs of interest. As proof of concept, a T7 phage display library was constructed using P51G-m4-CVN genes mutated at positions 41, 44, 52, 53, 56, 74, and 76 in binding Domain B. Five CV-N mutants were selected from the library and expressed in BL21(DE3) E. coli. Two of the mutants, SSDGLQQ-P51Gm4-CVN and AAGRLSK-P51Gm4-CVN, were sufficiently stable for characterization and were examined by CD, Tm, ELISA, and glycan array. Both proteins have CD minima at approximately 213 nm, indicating largely β-sheet structure, and have Tm values greater than 40°C. ELISA against gp120 and RNase B demonstrate both proteins' ability to bind high mannose glycans. To more specifically determine the binding specificity of each protein, AAGRLSK-P51Gm4-CVN, SSDGLQQ-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN were sent to the Consortium for Functional Glycomics (CFG) for glycan array analysis. AAGRLSK-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN, have identical specificities for high mannose glycans containing terminal Manα1-2Manα. SSDGLQQ-P51Gm4-CVN binds to terminal GlcNAcα1-4Gal motifs and a subgroup of high mannose glycans bound by P51G-m4-CVN. SSDGLQQ-wt-CVN was produced to restore anti-HIV activity and has a high nanomolar EC50 value compared to wt-CVN's low nanomolar activity. Overall, these experiments show that CV-N Domain B can be mutated and retain specificity identical to wt-CVN or acquire new glycan specificities. This first generation information can be used to produce glycan-specific lectins for a variety of applications.
ContributorsRuben, Melissa (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2013
Description
The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and immunoadjuvant together, and strong antibody response was demonstrated in vivo, highlighting the potential of DNA nanostructures to serve as a new platform for vaccine construction, and therefore a DNA scaffolded hapten vaccine is further constructed and tested for its antibody response. Taken together, my research demonstrated the potential of DNA nanostructure to serve as a general platform for immunological applications.
ContributorsLiu, Xiaowei (Author) / Liu, Yan (Thesis advisor) / Chang, Yung (Thesis advisor) / Yan, Hao (Committee member) / Allen, James (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2012
Description
Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available chemical modifications, and intrinsic nanoscale size makes DNA a suitable material for the construction of a plethora of nanostructures that can be used as scaffold to organize functional molecules with nanometer precision. This dissertation focuses on DNA-directed organization of metallic nanoparticles into well-defined, discrete structures and using them to study photonic interaction between fluorophore and metal particle. Presented here are a series of studies toward this goal. First, a novel and robust strategy of DNA functionalized silver nanoparticles (AgNPs) was developed and DNA functionalized AgNPs were employed for the organization of discrete well-defined dimeric and trimeric structures using a DNA triangular origami scaffold. Assembly of 1:1 silver nanoparticle and gold nanoparticle heterodimer has also been demonstrated using the same approach. Next, the triangular origami structures were used to co-assemble gold nanoparticles (AuNPs) and fluorophores to study the distance dependent and nanogap dependencies of the photonic interactions between them. These interactions were found to be consistent with the full electrodynamic simulations. Further, a gold nanorod (AuNR), an anisotropic nanoparticle was assembled into well-defined dimeric structures with predefined inter-rod angles. These dimeric structures exhibited unique optical properties compared to single AuNR that was consistent with the theoretical calculations. Fabrication of otherwise difficult to achieve 1:1 AuNP- AuNR hetero dimer, where the AuNP can be selectively placed at the end-on or side-on positions of anisotropic AuNR has also been shown. Finally, a click chemistry based approach was developed to organize sugar modified DNA on a particular arm of a DNA origami triangle and used them for site-selective immobilization of small AgNPs.
ContributorsPal, Suchetan (Author) / Liu, Yan (Thesis advisor) / Yan, Hao (Thesis advisor) / Lindsay, Stuart (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2012
Description
The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration.
ContributorsMaini, Rumit (Author) / Hecht, Sidney M. (Thesis advisor) / Gould, Ian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.
ContributorsBowen, Sara (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2010
Description
A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism (CD) spectroscopy was used to elucidate the role of Cu2+ in stabilizing SmbP structure against unfolding induced by decreased pH, increased temperature, and chemical denaturants. The significant stabilization effects of Cu2+ were demonstrated by the observation that Cu2+-SmbP remained fully folded under extreme environmental conditions, such as acidic pH, 96 °C, and 8 M urea. Also, it was shown that Cu2+ is able to induce the refolding of unfolded SmbP in acidic solutions. These findings imply that the coordination of Cu2+ to histidine residues is responsible for the stabilization effects. The crystal structure of SmbP without Cu2+ has been determined. However, attempts to crystallize Cu2+-SmbP have not been successful. In this study, multidimensional NMR experiments were conducted in order to gain additional information regarding the Cu2+-SmbP structure, in particular its metal binding sites. Unambiguous resonance assignments were successfully made. Cα secondary chemical shifts confirmed that SmbP has a four α-helical structure. A Cu2+-protein titration experiment monitored by NMR indicated a top-to-bottom, sequential metal binding pattern for SmbP. In addition, several bioinformatics tools were used to complement the experimental approach and identity of the ligands in Cu2+-binding sites in SmbP is proposed.
ContributorsYan, Qin (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010