Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
Browse
38 results
Search Results
Item Development and applications of codon scanning mutagenesis: A novel mutagenesis method that facilitates in-frame codon mutations(2009) Daggett, Kelly Anne; Cropp, Ashton; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The ability to create protein variants is a very valuable tool in biochemistry. Information about mechanistic roles of amino acid side chains, protein topology and binding can all be obtained. Methodologies to mutate proteins also allow for new catalytic activity to be achieved. While the routinely used methods to alter a protein sequence have proven to be useful, to some degree each of these methods requires some knowledge of protein structure to determine the site of mutation. Further, the routinely used methods also only allow for a specified site to be changed to a pre-determined residue (directed by oligonucleotides) or for multiple random sites to be changed to a non-specified residue. This dissertation focuses on the development of a method that allows for a new defined amino acid to replace a native amino acid at a random location within in the protein. To introduce mutations at random locations within a protein coding sequence, three steps need to be accomplished. First, the coding sequence needs to be randomly digested on both strands; second, three nucleotides (a codon) at the digestion site need to be removed; and last, a new specified codon inserted. This process results in the replacement of a random codon with the new defined codon. To direct a mutation at a random location, the unique properties of a transposase/transposon are used to create both the double strand break and removal of three nucleotides. The insertion of the new defined codon is introduced using a linker sequence that when inserted in the correct reading frame a selectable phenotype is produced. This process has been termed Codon Scanning Mutagenesis (CSM). The advantages of this method over current mutagenesis methods are (1) knowledge of structural information is not required, (2) oligonucleotides are not required to introduce the mutation and (3) the mutagenesis method allows for every amino acid to be mutated regardless of the DNA sequence. Further, this method allows for any natural and unnatural amino acid to be inserted at the mutation site, as well as the ability to create mutational mixtures or introduce multiple user defined mutations.Item DENSITY FUNCTIONAL CALCULATIONS OF BACKBONE 15N CHEMICAL SHIELDINGS IN PEPTIDES AND PROTEINS(2009) Cai, Ling; Fushman, David; Kosov, Daniel S; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation, we describe computational and theoretical study of backbone 15N chemical shieldings in peptides and proteins. Comprehensive density functional calculations have been performed on systems of different complexity, ranging from model dipeptides to real proteins and protein complexes. We begin with examining the effects of solvation, hydrogen bonding, backbone conformation, and the side chain identity on 15N chemical shielding in proteins by density functional calculations. N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) were used as model systems for this purpose. The conducting polarizable continuum model was employed to include the effect of solvent in the calculations. We show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard &beta-sheet and standard &alpha-helical conformations ranges from 0.8 ppm to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins. Having applied density functional calculation successfully to model peptides, we develop a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone 15N in a protein. We present the application to selected &alpha-helical and &beta-sheet residues of protein G. The role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field is analyzed. We show that the dipole moment of the &alpha-helix can cause significant deshielding of 15N; therefore, it needs to be considered when calculating 15N chemical shielding. We emphasize the importance of including interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. For &alpha-helical residues, chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data. We also performed computational modeling of the chemical shift perturbations occurring upon protein-protein or protein-ligand binding. We show that the chemical shift perturbations in ubiquitin upon dimer formation can be explained qualitatively through computation. This dissertation hence demonstrates that quantum chemical calculations can be successfully used to obtain a fundamental understanding of the relationship between chemical shielding and the surrounding protein environment for the elusive case of 15N and therefore enhance the role of 15N chemical shift measurements in the analysis of protein structure and dynamics.Item CATALYTIC FEATURES OF THE IODINE SALVAGING ENZYME IODOTYROSINE DEIODINASE(2009) McTamney, Patrick Michael; Rokita, Steven E.; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The need for iodide in biology is almost exclusively limited to its role in thyroid hormones, yet the recycling of thyroidal iodide is still critical for human health. The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from byproducts (mono– and diiodotyrosine, MIT and DIT) of thyroid hormone biosynthesis. The original proposal for the deiodination mechanism of IYD included a nucleophilic attack on the iodo group by an active site cysteine. Although this proposal had strong precedence, site–directed mutagenesis has now proven this wrong. Further investigation of the IYD mechanism required large scale protein expression and isolation. This was stymied by the lack of a convenient isolation system until a truncated and soluble version of wild–type IYD could be expressed in yeast and insect cells. Large scale isolation of this soluble enzyme derivative provided the necessary material for crystallographic studies that in turn resulted in a structure of IYD at 2.0 Å resolution. The structure verified IYD's assignment in the NAD(P)H oxidase/flavin reductase superfamily and showed that no cysteine residues were in the active site. Structures of IYD with bound MIT and DIT were also obtained and indicated that these substrates are sequestered within the active site by inducing helical structure in two otherwise disordered regions of the enzyme to form an active site lid. This lid confers substrate specificity and is critical in positioning substrate such that it stacks on the isoalloxazine of the flavin mononucleotide (FMN) cofactor. Further investigation identified 3–bromo and 3–chlorotyrosine as substrates for IYD, while 3–fluorotyrosine was not dehalogenated by IYD. These new substrates illustrate IYD's activity as a general dehalogenase and IYD's strong dehalogenating power. Mechanistic studies utilizing 5–deazaFMN, which is incapable of performing 1 electron processes, indicated that IYD dehalogenation occurs via two sequential 1 electron transfers from reduced FMN to substrate. Anaerobic single turnover assays and mechanistic precedence have led to a likely mechanism of dehalogenation for IYD involving substrate tautomerization followed by injection of an electron into the carbonyl of the keto intermediate which then facilitates dehalogenation.Item BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THREE DNA REPAIR ENZYMES IN DEINOCOCCUS RADIODURANS(2009) Cao, Zheng; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram positive bacterium Deinococcus radiodurans is able to withstand acute doses of gamma rays that can cause hundreds of double-strand breaks per genome. In proposed double-stand break repair pathways, however, some important enzymes, such as helicases and nucleases in the initiation step, have not been clearly identified yet. Interestingly, the common bacterial helicase/nuclease complex RecBCD or AddAB, which functions to produce a 3' ssDNA tail in double-strand break repair initiation step in other bacteria, is not found in D. radiodurans. As part of efforts to identify helicases involved in double-strand break repair, the D. radiodurans HelIV (encoded by locus DR1572, the helD gene) was characterized with both in vivo and in vitro methods. The helD gene is predicted to encode a helicase superfamily I protein. The helD mutant is moderately sensitive to methyl methanesulfonate and hydrogen peroxide but it is not sensitive to gamma rays, UV and mitomycin C. In biochemical assays, the full length HelIV exhibited DNA unwinding activity with a 5'-3' polarity whereas the truncated HelIV without N-terminal region had no detectable helicase activity. RecJ is the exonuclease in the RecF pathway, which is suggested to function at the initiation step in DSB repair in the absence of RecBCD. In the in vivo study, the D. radiodurans recJ gene (encoded by locus DR1126) cannot be completely removed from the chromosome, indicating the essential role of RecJ in cell growth. The heterozygous mutant displayed growth defect and higher sensitivity to gamma rays, hydrogen peroxide and UV compared to wild type D. radiodurans, suggesting an important role in DNA repair. The RecJ expressed in E. coli system was insoluble but can be purified via denaturation-refolding, and the refolded RecJ showed 5'-3' exonuclease activity. D. radiodurans has no RecB and RecC proteins, but it has a homologue of the RecD protein. We tested whether the D. radiodurans RecD protein could form a complex or make transient interactions with other proteins to perform more complicated functions. The RecD conjugated protein affinity column was used to attempt to identify cellular binding partners.Item Biochemical characterization of the Minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus(2009) Sakakibara, Nozomi; Julin, Douglas; Kelman, Zvi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA replication requires coordination of numerous proteins to duplicate genetic information in a precise and timely manner. One of the key players in replication is the replicative helicase that unwinds the duplex DNA to provide the single-stranded template for the DNA polymerases. Minichromosome maintenance (MCM) protein is the replicative helicase in archaea. This dissertation focuses on the MCM helicase from the euryarchaeon Methanothermobacter thermautotrophicus (Mth). Archaeal MCM proteins can be divided into two major parts, the N terminal and C terminal domains. The N terminal domain is essential for DNA binding and multimerization, while the C-terminus contains the catalytic domains. The objective of this dissertation is to elucidate the mechanism by which the N terminal domain communicates with the catalytic domain to facilitate helicase activity. To address this question, two approaches were taken. One approach identified conserved residues found in the N terminus and investigated their properties using various biochemical and biophysical methods. By analyzing several proteins with mutations in the conserved residues, a loop that is essential for MCM helicase activity was identified. The study suggests that the loop is involved in coupling the N-terminal DNA binding function and the catalytic activity of the AAA+ domain. Some other conserved residues, however, did not directly affect the MCM helicase activity but showed differences in biochemical properties suggesting that they may play a role in maintaining the structural integrity of the MCM helicase. Another approach determined the differences in thermal stability of the MCM protein in the presence of various cofactors and DNA substrates. The study shows that the protein has two unfolding transitions when ATP and the DNA are present, while non-hydrolyzable ATP results in one transition. This study suggests possible conformational changes arising from decoupling of two domains that occur during the ATP hydrolysis in the presence of DNA. Furthermore, both DNA binding function by the N terminal domain and ATP binding by the catalytic domain are essential for the change.Item Investigation into the Driving Force Dependence of Excess Electron Transport in Duplex DNA(2009) Campbell, Neil Peter; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The purpose of this dissertation is to investigate the driving force dependence of excess electron transport in deoxyribonucleic acid (DNA) using napthyl amines as electron donors. The ability of DNA to act as a pathway for the migration of charge was first proposed in 1963 by Elgy and Spivey. Since then, investigation of two complementary processes, hole transport and excess electron transport, have been studied. Of these processes research has focused mostly on hole transport. Hole transport has been studied for several decades. As such, the four fundamental parameters affecting the processes have been elucidated: the distance dependence has been found to be weak, G/C sequences have been found to allow for more efficient hole transport, migration from the 3' to 5' direction is more efficient, and a driving force dependence on the efficiency of hole transport has been found. Only recently has attention turned to the determination of the fundamental parameters affecting excess electron transport. Investigations to date have determined that there is a weak distance dependence on excess electron transport, A/T sequences allow more efficient transport, and excess electron transport is more efficient when migrating from the 5' to 3' end of DNA. The one parameter affecting excess electron transport that has not been investigated is the driving force dependence. To test for driving force dependence, napthyl amines were screened for their ability to initiate charge transfer by reductive electron donation using an assay based on the photoinduced reduction and subsequent scission of duplex DNA containing a 5-bromo-2'-deoxyuridine (BrU) residue and an abasic site. Each compound had varying reducing potentials (driving forces), which allowed investigation into the driving force dependence of excess electron transport. Six compounds (1,5-diaminonapthalene, N1-methyl-1,5-diaminonathalene, N1,N5-dimethyl-1,5-diaminonapthalene, N1,N1-dimethyl-1,5-diaminonapthalene, N1,N1,N5-trimethyl-1,5-diaminonapthalene, N1,N1,N5,N5-tetramethyl-1,5-daiminonapthalene) were screened under both aerobic and anaerobic conditions, and found to initiate charge transfer. No correlation between the reduction potential of the compounds (driving force) and the rate of strand scission was seen. Subsequently, the oligonucleotide conjugates of two of the compounds, 1,5-diaminonapthalene and N1,N1,N5,N5-tetramethyl-1,5-diaminonapthalene, were prepared and studied to determine if a driving force dependence on excess electron transport exists when the compounds are covalently attached to the DNA. 1,5-diaminonapthalene and N1,N1,N5,N5-tetramethyl-1,5-diaminonapthalene were chosen as they showed the greatest difference in their reducing potentials. The conjugates showed no difference in the rate of excess electron transport, thus indicating there is not a driving force dependence on excess electron transport in DNA, at least using these compounds and in this system.Item SCAFFOLDING-MEDIATED VIRUS ASSEMBLY: VISUALIZATION AND CHARACTERIZATION OF BACTERIOPHAGE T7 SCAFFOLDING PROTEIN(2009) Smith, Charles Stewart; Beckett, Dorothy; Steven, Alasdair C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In bacteriophage T7 as well as many other dsDNA phages and some animal viruses, scaffolding protein is essential for the accurate formation of the metastable precursor particle (prohead) and thus it is a vital aspect of the viral infection cycle. When purified by anion exchange and gel filtration chromatography, the T7 scaffolding protein (gp9) exists as an extended monomer in solution. These monomers of gp9 associate with other scaffolding monomers to form the extended filaments in T7 procapsids visualized by electron tomography. These filaments interact with the negatively charged inner surface of the procapsid at unique sites, probably via the extended positively charged C-terminus of gp9. Scaffolding protein, via these interactions, facilitates isometric capsid assembly by helping to define the proper curvature of the viral capsid. Consequently, scaffolding-mediated viral assembly does not require a rigid structural network, nor does it require a stoichiometric amount of gp9 to be present in each procapsid. The observed flexibility of the scaffolding network, association of scaffolding filaments with the core connector complex, as well as the variable copy number of gp9 per particle suggests an assembly mechanism where capsid formation is nucleated around the core/connector complex. In such a mechanism there is more than one path to a single end - production of the correctly formed prohead particles that are required for T7 phage maturation.Item SINGLE MOLECULE FLUORESCENCE INVESTIGATION OF PROTEIN FOLDING(2008-09-30) liu, jianwei; Munoz, Victor; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In addition to the well known two-state folding scenario, the energy landscape theory of protein folding predicts the possibility of downhill folding under native conditions. This intriguing prediction was extended by Victor Muñoz and coworkers to include global downhill folding. i.e. a barrierless free energy surface and unimodal conformational distributions at all degrees of unfolding stress. A small protein, BBL, has been shown to follow this behavior as evidenced from experiments and simulations. However, the identification of BBL as a global downhill folder has raised a significant amount of controversy with some groups claiming that it still folds in a two-state fashion. The objective of this thesis is to characterize the conformational distribution of BBL using single molecule Förster resonance energy transfer (SM-FRET) to obtain direct evidence for the downhill folding in BBL. We carried out SM-FRET measurements at 279 K to slow down the protein dynamics to 150 μs thus enabling the use of a 50 μs binning time (the short binning time being a first in SM measurements). By optimizing the microscope system setup and employing a novel Trolox-cysteamine fluorophore protection system, we obtained sufficient signal to construct reliable 50 μs SM-FRET histograms. The data show clear unimodal conformational distributions at varying denaturant concentrations thus demonstrating the downhill folding nature of BBL. Further SM-FRET measurements on a two-state folder, α-spectrin SH3 produced bimodal histograms indicating that our experimental setup works well and that the unimodal distributions of BBL are not due to instrumental errors. The comparison of ensemble FRET measurements on labeled proteins (both BBL and α-spectrin SH3) with CD measurements on the corresponding unlabeled proteins shows that the fluorophores do not affect the protein stability. We also simulated the expected histograms if BBL were a two-state folder using Szabo's photon statistics theory of SM-FRET. The two-state simulation results are inconsistent with the experimental histograms even under very conservative assumptions about BBL's relaxation time. Therefore, all the control experiments and simulations exclude any possible artifacts, which shows our results are quite robust. Additionally, we estimated the relaxation time of BBL from the histogram width analysis to be consistent with independent kinetic measurements.Item Mutational Analysis of the Downhill Folding Protein gpW: Towards Tuning Stability of a Molecular Rheostat Candidate(2008-09-24) Fung, Adam; Munoz, Victor; Beckett, Dorothy; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A popular convention derived from early experimental evidence of single-domain proteins pointed towards a common mechanism of achieving their native three-dimensional structure. Concomitantly, protein function has been linked to the simple two-state approach wherein the folded state is associated with its biologically active conformation. The more recent discovery of downhill protein folding has provided a sharp contrast to this simple treatment. A catalog of qualitative signatures has been developed to distinguish between these different folding mechanisms at the two extremes (downhill and two-state). Additionally, the introduction of physical models to measure the protein folding ensemble allows the direct measurement of both the thermodynamic and kinetic barrier heights. The end result of such a quantitative approach is a more distinct partition separating the two scenarios. This methodology has been applied to the bacteriophage lambda protein gpW (gene product W) where a clear assessment of its folding behavior has been obtained. GpW is involved at the connector region at which assembly of bacteriophage heads and tails occurs. Without gpW, infectious virions are incapable of forming. This protein is also suspected of having a role in DNA binding and packaging. Contrary to its expected two-state behavior, gpW folds with a marginal barrier (< 3 RT) as determined through an examination of the thermodynamic and kinetic behaviors. Possessing a novel fold and being an independently folding protein, gpW represents the first experimentally characterized downhill folder that is not a domain of a larger complex and is known to perform a specific function. This is a clear diversion from the standpoint of a single macrostate being responsible for function and places a significant emphasis on investigating the functional role of downhill folding. With the function of performing multiple duties, gpW is an excellent candidate for functional studies of downhill folding.Item TARGETING BIOMARKERS VIA CITP-BASED SELECTIVE PROTEOME ENRICHMENT(2008-06-30) Fang, Xueping; Lee, Cheng S; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Besides the complexity of protein samples, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. To achieve comprehensive proteome analysis including the identification of low abundance proteins, this project aims to develop and demonstrate a capillary isotachophoresis (CITP)-based proteome platform, capable of providing selective analyte enrichment and extremely high resolving power toward complex protein mixtures. In contrast to universally enriching all proteins by a similar degree, the result of the CITP stacking process is that major components may be diluted, but trace compounds are concentrated. By employing combined CITP/nano-reversed phase liquid chromatography (nano-RPLC) separations, a total of 6,112 fully tryptic peptides are sequenced by electrospray ionization mass spectrometry (ESI-MS), leading to the identification of 1,479 distinct human SwissProt protein entries from a single proteome sample of whole unstimulated human saliva. By comparing with capillary isoelectric focusing as another electrokinetics-based stacking approach, CITP not only offers a broad field of application, but also is less prone to protein/peptide precipitation during the analysis. The CITP-based proteome platform is further employed for the analysis of protein expression within synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP separation is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, the collective proteome datasets yield the identification of 2,191 distinct mitochondrial protein entries, corresponding to 76% coverage of the MitoP2-database reference set. Comparisons among CITP and multidimensional liquid chromatography techniques are conducted using a single processed protein digest from brain cancer stem cells, identical second dimension separation (nano-RPLC) and ESI-MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications, analytical reproducibility of the CITP proteome platform is determined by a Pearson R2 value of 0.98 and a coefficient of variation of 15% across all proteins quantified.