Cell Biology & Molecular Genetics Theses and Dissertations
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Item A Comparative Analysis of the Binding Affinity of HIV-1 Reverse Transcriptase to DNA vs. RNA Substrates(2010) Olimpo, Jeffrey T.; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3´ recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2´- hydroxyls on pivotal RNA bases with 2´-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, this may aid in degradation of RNA fragments that remain after DNA synthesis.Item COMPARING LEVELS OF NDH-1 DEHYDROGENASE ACTIVITY IN DIFFERENT MYCOBACTERIAL SPECIES.(2008) Azogue, Sharon; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The nuoG gene of Mycobacterium tuberculosis (Mtb) has the ability to inhibit host cell apoptosis. This ability is a virulence factor and does not exist in facultative pathogenic and non-pathogenic mycobacterial species. NuoG is part of the NDH-1 complex, and this study addressed the potential link between the role of NuoG in apoptosis inhibition and the biochemical activity of the NDH-1 complex. Different mycobacterial species were tested for their NDH-1 activities. Among the bacteria tested were bacteria transformed with the Mtb nuoG plasmid, or with the almost entire NDH-1 coding region. Surprisingly, the levels of NDH-1 activity did not correlate with apoptosis levels, suggesting a potential independent, novel mechanism by which NuoG inhibits host cell apoptosis.Item Binding Interactions in the Bacterial Chemotaxis Signal Transduction Pathway(2008-12-08) Eaton, Anna Kolesar; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.Item Energetics of Drug Interactions(2008-11-26) Todorova, Niya Ancheva; Kelman, Zvi; Schwarz, Frederick P.; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The goal of our research is to determine in terms of thermodynamic change of state functions the effects of experimental factors, such as water, mutagenesis, or the presence of a second substrate on the energetics of drug-inhibitor binding interactions. The binding of non-steroidal anti-inflammatory drugs within the rigid cavities of cyclodextrins was investigated by titration calorimetry and spectrofluorimetry. Loss of bulk water structure upon drug binding in the smaller hydrophobic β-cyclodextrin cavity results in an increase in the binding entropy, while restriction of the configurations of the drug in the cavity decreases the binding entropy. This restriction in the hydrophobic β-cyclodextrin cavity enhances the binding enthalpies so that the β-cyclodextrin binding reactions are enthalpy-driven. In the larger γ-cyclodextrin cavity, water is retained so that, not only are the interactions between the drug and the cavity reduced, there is an increase in the drug configurations resulting in increases in the binding entropies and the binding reactions become entropically-driven. These binding reactions also manifest enthalpy-entropy compensation where changes in the binding enthalpies are compensated by changes in the binding entropies. In drug binding to the more flexible p38α MAP kinase mutants, a single-point C→S mutation distal from the binding site, changes the interaction between the N- and C-terminal structural domains of the kinase as evident in differential scanning calorimetry. Calorimetric results show that drug-inhibitor binding affinities to kinase increase with size of the drugs since the binding reactions are all enthalpically-driven. Drug-inhibitors binding to trimeric human purine nucleoside phosphorylase were investigated by calorimetry in the presence of its second substrate, inorganic phosphate (Pi). Increasing concentrations of Pi modulates the driving-nature of the binding reaction, so that the acyclovir binding almost exclusively to the purine substrate binding site becomes more entropically-driven, while the binding reactions of ganciclovir and 9-benzylguanine interacting also with the adjacent Pi substrate site become more enthalpically-driven. A novel calorimetric enzyme activity assay at the low dissociation concentrations of the phosphorylase show an increase in the enzyme activity at low Pi concentrations, but also a decrease in the 9-benzylguanine binding affinity since this drug also interacts with an adjacent subunit.Item The Structural Basis for Function of the Escherichia coli Mechanosensitive Channel of Small Conductance, MscS(2007-04-26) Akitake, Bradley Chun-Yee; Sukharev, Sergei; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The 'small' mechanosensitive channel, MscS, resides in cytoplasmic membranes of most free-living bacteria. MscS is gated directly by membrane tension and functions as an osmolyte release valve in bacterial turgor regulation. In contrast to previously studied MscL, which is a strictly prokaryotic molecule, MscS homologs are found in eukaryotes increasing the value of this channel as a general model for gating by membrane stretch. Presented here are the results of three studies aimed at characterizing the structural basis for function of Escherichia coli MscS. In study one, we provide the first electrophysiological characterization of the wild-type channel in its native membrane free of other mechanosensitive channels. It is, to date, the most complete description the gating cycle specifying the kinetic scheme and dependencies of major rates on tension and voltage. Study two represents a collaborative effort to probe the strength of intersubunit contacts in the homo-heptameric MscS channel. In patch-clamp experiments we show that the dissociating effects of TFE alter MscS gating in a manner that provides significant insight into the mechanics of channel inactivation. In the final study our research group utilized a novel extrapolated motion technique to explore the conformational pathways of the MscS functional cycle. Guided by these new models, channel mutants were generated to alter helical propensity along the pore lining TM3 helix. Patch-clamp analysis revealed a vivid picture of the functioning MscS in which these TM3 domains provide a structural frame for the open channel. Dynamic collapse of these 'struts' at flexible points along TM3 modulates transitions from the open state to the inactivated and closed states. My contributions to these studies have allowed for (1) refinement of the MscS functional cycle including identification of a new desensitized state; (2) determination of the physical parameters and spatial scales of channel opening, closing and inactivation; and (3) identification of key hinge elements, residing in TM3, that along with membrane tension serve to modulate the functional cycle of MscS. These findings have led to a better understanding of the biophysical principles that underlie mechanotransduction and provide insights into the larger family of mechanically activated phenomena.Item Structural characterization of metal and DNA binding to DREAM protein, a calcium sensing transcriptional repressor in pain modulation(2006-09-29) Valiveti, Aswani Kumar; Ames, James B; Julin, Douglas; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DREAM (Downstream Regulatory Element Antagonistic Modulator) is the first reported calcium binding protein that directly binds to DNA and affects gene expression. It belongs to recoverin sub-family of neuronal calcium sensing proteins that form part of EF- hand super-family. Knockout mice, devoid of DREAM protein, exhibit ongoing analgesia due to upregulated expression of the prodynorphin gene. DREAM binds to a specific DNA silencing element in the prodynorphin promoter called Downstream Regulatory Element (DRE) and serves as a transcriptional repressor under basal resting conditions. Neuronal stimulation leads to a rise in nuclear calcium, causing Ca++-bound DREAM to dissociate from DNA and result in derepression of prodynorphin expression. Thus, DREAM may serve as a potentially specific target for effective pain management. Understanding the structural determinants of metal binding and DNA recognition by DREAM may help in the future rational design of analgesic drugs. In this thesis, the calcium and magnesium binding properties of DREAM were studied by performing isothermal titration calorimetry (ITC) analysis on wild-type DREAM protein and mutants that have each of the functional EF hands disabled for calcium binding. Our results show that DREAM binds two Ca++ ions and one Mg++ ion with high affinity. Mg++-bound DREAM is stable as a monomer and Ca++-bound DREAM exists as a dimer. Calcium binding stabilizes the protein structure and promotes dimerization to control calcium-sensitive recognition of DNA targets. Next, we attempted to map the DNA binding residues in DREAM and its oligomerization interface using biophysical and functional characterization of chimeric protein constructs generated by swapping specific EF-hand motifs of DREAM protein with those of recoverin. Nuclear magnetic resonance (NMR), gel-filtration and electrophoretic mobility shift assays were employed for this purpose. The results from this study suggest that both the amino and carboxy terminal halves of the protein are important for DNA binding and dimerization of DREAM protein. All four EF-hands in DREAM appear to participate in the recognition of DNA.Item In Vitro Synthesis of Long Reverse Transcription Products from Genomic RNA of Human Immunodeficiency Virus(2006-04-25) Anthony, Reshma Merin; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The retroviral reverse transcription reaction normally occurs in capsid-like structures in the cytoplasm of infected cells. Reverse transcription can also be carried out in vitro in totally reconstituted reactions with purified enzymes and model RNA templates. However, in this case fully synthesized DNAs are rarely generated from genomic RNA. This could be because the capsid creates an extremely concentrated and specific environment that cannot be completely reproduced in vitro. An in vitro system that closely mimics replication and that can be easily manipulated would enhance our understanding of the replication process. In this thesis report, in vitro reaction conditions that allowed efficient synthesis of DNA products up to 4 kb from genomic RNA segments of Human Immunodeficiency Virus (HIV) were generated. The reactions required high amounts of HIV reverse transcriptase enzyme (RT) and nucleocapsid protein (NC) sufficient to completely coat the RNA template in the reaction. Synthesis of long DNA products required the formation of high molecular weight aggregates with nucleic acids, RT and NC. Removal of the dimerization region did not affect synthesis of long DNA products in vitro. Processivity of RT does not play a role in the synthesis of long DNA products. NC finger mutants lacking either finger or with the finger positions switched were all effective in synthesizing long DNA products suggesting that the aggregation/condensation activity but not the unwinding activity of NC is required for the synthesis of long DNAs in vitro. These results taken together, we propose that high molecular weight aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC into a smaller area, thereby mimicking the role of the capsid environment within the host cell. In addition, strand transfer assays indicate that strand transfer is the molecular mechanism involved in the synthesis of long DNAs and the rate of transfer (cross-over events per nucleotide synthesized) is higher than that found in tissue culture-based recombination assays. An in vitro system that closely mimics what occurs in the cell could be used to screen inhibitors on RT, NC and recombination.Item STRUCTURAL AND FUNCTIONAL ANALYSIS OF DNA REPLICATION INITIATION PROTEINS FROM THE ARCHAEON METHANOTHERMOBACTER THERMAUTOTROPHICUS(2005-12-01) Kasiviswanathan, Rajesh; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The faithful duplication of the chromosome requires the combined efforts of numerous proteins. Cdc6 and MCM are two such proteins involved in the initiation of DNA replication. The genome of the euryarchaeon Methanothermobacter thermautotrophicus contains one MCM and two Cdc6 homologues (Cdc6-1 and -2). While MCM is the replicative helicase that unwind the duplex DNA to provide single-stranded DNA substrate for the replicative polymerases, the Cdc6 proteins are presumed to function in origin recognition and helicase assembly at the origin. This thesis elucidates the structure, function and regulation of these archaeal initiation proteins. The M. thermautotrophicus MCM helicase is a dumb-bell shaped double hexamer. Each monomer can be divided into two portions. The C-terminal catalytic region contains the ATP binding and hydrolysis sites essential for helicase activity. This thesis concentrates its efforts to determine the functional role of the N-terminal region. Using a variety of biochemical approaches it was found that the N-terminal portion of MCM is involved in hexamer/dodecamer formation. The study also identified two structural features at the N-terminus, the zinc- and the beta-finger motifs, essential for DNA binding, which in turn is essential for helicase activity. In addition, the N-terminal portion of MCM interacts with both Cdc6 proteins. The role of the Cdc6-1 and -2 proteins in origin recognition and helicase loading was also elucidated. The results presented in this thesis show that Cdc6-1 has binding specificity to origin DNA sequences suggesting a role for the protein in origin recognition. While both Cdc6 proteins interact with the MCM helicase, Cdc6-2 exhibited tighter binding compared to Cdc6-1 suggesting a role for Cdc6-2 in helicase loading. Summarizing the observations of this study, a model for the replication initiation process in M. thermautotrophicus has been proposed, outlining separate role for the two Cdc6 proteins, Cdc6-1 in origin recognition and Cdc6-2 in MCM helicase assembly at the origin.Item Patterns of reactivity of lantibiotics subtilin and nisin with molecular targets in Bacillus cereus and Bacillus subtilis 168(2005-01-21) Kuntumalla, Srilatha; Hansen, J Norman; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: PATTERNS OF REACTIVITY OF LANTIBIOTICS SUBTILIN AND NISIN WITH MOLECULAR TARGETS IN Bacillus cereus AND Bacillus subtilis 168 Srilatha Kuntumalla, Doctor of Philosophy, 2005 Dissertation Directed By: Professor J. Norman HansenDepartment of Chemistry and Biochemistry Subtilin and nisin belong to a unique class of antibiotics called lantibiotics that contain unusual dehydro and lanthionine amino acid residues. The gene-encoded antimicrobial peptides subtilin and nisin exhibit bactericidal effects against several Gram-positive bacteria and also inhibit bacterial spore outgrowth. Subtilin and nisin are structural analogs and possess similar mechanisms of antimicrobial action. Although nisin is very stable, subtilin previously isolated was highly unstable with loss of biological activity observed during storage. Subtilin isolated in this work using hydrophobic interaction chromatography was very stable, with biological activity retained for at least a few months after isolation. The possibility that specificity of subtilin and nisin towards sensitive Gram-positive bacteria is due to interaction of these lantibiotics with specific target proteins in susceptible bacteria was explored in this work. Phage display experiments performed to detect peptides interacting with subtilin identified a 12-mer peptide with a KTTLL motif found in ATP binding proteins such as ABC transporters and protein synthesis initiation factor IF-2 (~78 kDa). Binding of subtilin to specific ABC transporters in bacterial cell membrane would contribute to its specificity. Binding of subtilin to IF-2 would result in inhibition of protein synthesis suggesting an alternative mechanism of action for subtilin. Experiments performed to determine the nature of interaction of subtilin and nisin with bacterial cellular proteins detected both covalent and non-covalent interactions. The covalent interactions between bacterial proteins and subtilin or nisin were stable on boiling in SDS and analyzing by SDS-PAGE. These stable covalent adducts indicated that the electrophilic dehydro residues of subtilin and nisin were probably involved in covalent attachment with specific nucleophilic groups in bacterial protein targets. Covalent attachment of an antibiotic to its bacterial target has been previously observed with only a few antibiotics. Sites of nisin attachment to bacterial spores as visualized by electron microscopy showed nisin binds to highly localized regions on spore surfaces. Attempts to identify bacterial protein targets of subtilin and nisin using monomeric avidin and anti-FITC columns, respectively, resulted in isolation of proteins in ~70-80 kDa range. Further characterization of these proteins should help in understanding the specificity and antimicrobial mechanism of action of nisin and subtilin.Item Effect of Transcriptional Parameters on the Folding of the Tetrahymena Group I Intron(2004-01-29) Koduvayur, Sujatha P; Woodson, Sarah A; Julin, Douglas; Molecular and Cell BiologyDifferential elongation rates and pausing patters of polymerases are known to affect co-transcriptional folding of RNAs. The mechanism of coupling is not well studied. This study evaluates whether these factors contribute to the 20-50 fold splicing enhancement seen in vivo for the Tetrahymena group I intron. The splicing rates of T7 and Escherichia coli (E. coli) RNA polymerase (RNAP) transcripts were compared in vitro and in E. coli. T7 RNAP is a rapid and highly processive polymerase, while E. coli RNAP elongates transcripts more slowly. In a collaborative study with Scott Jackson, the splicing of transcripts of eukaryotic polymerase I and II (pol I and pol II) were compared in Saccharomyces cerevisiae. As the stability of nascent structures can be altered by changing the sequence of transcribed RNA, I have also studied the effect of rRNA exons on the folding of this RNA by comparing the splicing rates of pre-RNAs with E. coli and Tetrahymena thermophila domain IV rRNA exons in vitro and E. coli. In yeast, S. cerevisiae and T. thermophila rRNA, and GFP mRNA exon sequences were compared. My E. coli and in vitro comparative study reveals that co-transcriptional folding is the reason for the rate enhancement seen in vivo for this RNA. For E. coli RNAP transcripts this is effected by the template-dependant, site-specific pausing of the polymerase along the template. For the pre-RNA with Tetrahymena rRNA exons this is effected by the low stability of nascent structures that enable rapid rearrangement of non-native structures into native ones. In yeast, we find that both the polymerase and the RNA processing events affect folding of this intron. Pol I transcripts splice 10-fold better than their pol II counterparts and mRNA processing events retard splicing of short pol II transcripts by 10-fold. Moreover, in yeast, only mutations that increase the fraction of misfolded intermediates are rescued but not those that destabilize the native structure. This folding facilitation is however not dependant on the presence of longer rRNA exons in yeast as it is in E. coli. This might be indicative of a different folding facilitatory mechanism in yeast from that seen in E. coli.