Chemistry & Biochemistry Theses and Dissertations
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Item THERMODYNAMIC PROPERTIES OF THE UNFOLDED ENSEMBLE OF PROTEINS(2010) DESAI, TANAY MAHESH; MUNOZ, VICTOR; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A random coil, whose size is determined by its excluded volume, and net energetic interactions with its environment, has served as a representation of the unfolded ensemble of proteins. The work in this thesis involves equilibrium, nuclear magnetic resonance and time-resolved kinetics spectroscopic studies on the unfolded ensemble of BBL, a globally downhill folding 40-residue protein involved the Krebs cycle of E. coli, in its acid-denatured state, and on a sequence-randomized version of this protein. The effect of variability in thermodynamic conditions, such as temperature and the presence of added chaotropes or kosmotropes, on the equilibrium properties and reconfiguration dynamics of the unfolded state, have been deduced in the absence of competition with folding events at low pH. The unfolded ensemble experiences expansion and collapse to varying degrees in response to changes in these conditions. Individual interactions of residues of the protein with the solvent and the cosolvent (direct interactions), and the properties of the solution itself (indirect interactions) are together critical to the unfolded chain's properties and have been quantitatively estimated. Unfolded, protonated BBL can be refolded by tuning the properties of the solvent by addition of kosmotropic salts. Electrostatic interactions turn out to be essential for folding cooperativity, while solvent-mediated changes in the hydrophobic effect can promote structure formation but cannot induce long-range thermodynamic connectivity in the protein. The effect of sequence on the properties of heteropolymers is also tested with a randomized version of BBL's sequence. Chain radii of gyration, and the degree and rate of hydrophobic collapse depend on the composition of the sequence, viz. hydrophilic versus hydrophobic content. However, the ability to maximize stabilizing interactions and adopt compact conformations is more evident in naturally selected protein sequences versus designed heteropolymers. Chain reconfiguration of unfolded BBL takes place in ∼1/(100 ns), in agreement with theoretical estimates of homopolymer collapse rates. The refolding dynamics of salt-refolded BBL in the range of 1/(6 μs) at 320 K, emerge as being independent of the degree of folding or protonation of the chain, a result in keeping with the description of dynamics in BBL as oscillations in a single, smooth harmonic potential well, which only varies in its position and curvature with varying thermodynamic conditions.Item Top-Down Analysis of Bacterial Proteins by High-Resolution Mass Spectrometry(2010) Wynne, Colin Michael; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the biodefense and medical diagnostic fields, MALDI mass spectrometry-based systems are used for rapid characterization of microorganisms generally by detecting and discriminating the highly abundant protein mass-to-charge peaks. It is important that these peaks eventually are identified, but few bacteria have publicly available, annotated genome or proteome from which this identification can be made. This dissertation proposes a method of top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools to identify proteins from bacteria with unavailable genome sequences by comparison to protein sequences from closely-related microorganisms. Once these proteins are identified and a link between the unknown target bacteria and the annotated related bacteria is established, phylogenetic trees can be constructed to characterize where the target bacteria relates to other members of the same phylogenetic family. First, the top-down proteomic approach using an Orbitrap mass analyzer is tested using a well known, well studied single protein. After this is demonstrated to be successful, the approach is demonstrated on a bacterium without a sequenced genome, only matching proteins from other organisms which are thought to have 100% homology with the proteins studied by the top-down approach. Finally, the proposed method is changed slightly to be more inclusive and the proteins from two other bacteria without publicly available genomes or proteomes are matched to known proteins that differ in mass and may not be 100% homologous to the proteins of the studied bacteria. This more inclusive method is shown to also be successful in phylogenetically characterizing the bacteria lacking sequence information. Furthermore, some of the mass differences are localized to a small window of amino acids and proposed changes are made that increase confidence in identification while lowering the mass difference between the studied protein and the matched, homologous, known protein.Item Kinetic and Structual Characterization of Glutamine-Dependent NAD Synthetases(2010) Resto, Melissa; Gerratana, Barbara; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Multifunctional enzymes catalyzing successive reactions have evolved several mechanisms for the transport of intermediates between active sites. One mechanism, substrate channeling, allows the transport of the intermediate without releasing it into the solvent. Members of the glutamine amidotransferase (GAT) family often utilize substrate channeling for the transport of intermediates. GAT enzymes hydrolyze glutamine to ammonia, which is transported to an acceptor domain preventing wasteful hydrolysis of glutamine and increasing the efficiency of the reaction. Many GAT enzymes utilize molecular tunnels to shuttle ammonia between active sites. Often GAT enzymes synchronize the active site through conformational changes that occur during catalysis. Glutamine-dependent NAD synthetases are GAT enzymes and catalyze the last step in the biosynthesis of NAD, utilizing nicotinic acid adenine dinucleotide (NaAD), ATP and glutamine. Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetase from Mycobacterium tuberculosis (NAD synthetaseTB) revealed a substrate channeling mechanism for ammonia transport and tight coordination of the active sites resulting in an enzyme that is highly efficient in the use of glutamine. The crystal structure of NAD synthetaseTB has revealed a 40 Å tunnel that connects the active sites and is postulated to play a role in the synchronized activities. Several regions of the enzyme were identified that may be important for regulation, such as the YRE loop which contacts the glutamine active site and key regions of the tunnel. Mutations of tunnel residues, such as D656A, show that interruption of important interactions can result in compromise in transfer of ammonia or active site communication. Phylogenetic analysis revealed that glutamine-dependent NAD synthetases have different levels of regulation. Three groups of enzymes were identified represented by NAD synthetase from M. tuberculosis, S. cerevisiae (NAD synthetaseYeast) and Thermotoga maritima (NAD synthetaseTM). Steady-state kinetic characterizations and stoichiometric analysis of NAD synthetaseTM has revealed a compromised coordination of the active sites compared to the highly synchronized NAD synthetaseTB and the moderate synchronization of NAD synthetaseYeast. Sequence alignment of these groups has allowed identification of residues that line the tunnel that may be responsible for the differences observed in active site coordination and are, therefore, important for active site communication.Item Kinetic Characterization and Domain Analysis of the helicase RecD2 from Deinococcus radiodurans(2010) Shadrick, William Robert; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gram positive bacterium D. radiodurans is known for its extreme resistance to radiation and an extraordinary ability to reconstitute its genome after sustaining large numbers of double strand breaks (DSB's). Genome analysis does not immediately reveal a biochemical basis for this incredible DNA repair ability. In E. coli, DSB's are mainly repaired through the RecBCD pathway via homologous recombination. The D. radiodurans genome contains no known homologues of RecB or RecC, but sequence analysis has identified a homologue of RecD, termed RecD2. The function of RecD2 in D. radiodurans is unknown, as RecD elsewhere has only been found as a component of the RecBCD complex. Our research has focused on biochemical characterization of RecD2. Previous work in our lab established that RecD2 is a DNA helicase with limited processivity and a preference for forked substrates. We have studied the unwinding mechanism of the enzyme, as measured by rates of DNA unwinding and behavior on various substrates. Reactions conducted under single turnover conditions have allowed us to determine the processivity and the step size of RecD2. RecD2 pre-bound to dsDNA substrate is capable of unwinding 12 bp, but not 20 bp, when excess ssDNA is added to prevent rebinding of enzyme to substrate. Unwinding of the 12 bp substrate under single turnover conditions could be modeled using a two step mechanism, with kunw = 5.5 s-1 and dissociation from partially unwound substrate koff = 1.9 s-1. Results derived from these rate constants indicate an unwinding rate of 15-20 bp/ sec, with relatively low processivity (P = 0.74). Glutaraldehyde cross-linking showed formation of multimers of RecD2 in the absence of DNA, but this was not detectable by size exclusion chromatography. We were able to separate the N-terminal region from the helicase core of RecD2 using limited proteolysis. It was not possible to characterize the C-terminal helicase domain due to its low solubility upon overexpression in E. coli.Item The Development of New Tools for the Investigation of Protein Function Using Photo-Reactive Unnatural Amino Acids(2010) Wilkins, Bryan Jason; Cropp, Ashton; Gerratana, Barbara; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Reported here is the direct synthesis and application of unnatural amino acids for the development of exploratory tools for protein studies. This work takes advantage of an expanded genetic code to extract a more precise chemical understanding of protein function with novel additions to the unnatural amino acid catalogue, as well as the expansion of techniques with previously developed compounds. The photochemical crosslinker, [D11]-p-benzoylphenylalanine (pBpa), is synthesized for isotopic labeling in proteins. When [D11]-pBpa is co-incorporated into protein with [D0]-¬pBpa it is a mass spectral tool for rapid and conclusive identification of crosslinked fragments. Following enzymatic digestion the fingerprint of M, M+ 11 is readily identified allowing for rapid peak identification and the determined site of crosslink formation with single amino acid accuracy. In a means to extract a level of spatiotemporal control over fluorescent labeling of protein, the photo-protected unnatural amino acid, o-nitrobenzyl cysteine (ONBC), is introduced to a small amino acid tag sequence CCPGCC. This tag is required and specifically binds the pro-fluorescent compound 5-bis(1,3,2-dithiasolan-2-yl)fluorescein (FlAsH). This work takes advantage of the inability of FlAsH to bind the cysteine-tag motif in the presence of an ONBC mutation. The photo-protected amino acid is deprotected with light, affording natural cysteine and the successful binding of FlasH to the tetracysteine tag only following ultraviolet irradiation. Finally, fluorinated tyrosine derivatives are synthetically modified to contain photo-protecting groups, which act as a disguise during unnatural amino acid mutagenesis techniques. Fluorinated tyrosines are recognized by endogenous tyrosyl-tRNA synthetases and incorporated globally throughout a protein at tyrosine positions. To circumvent this problem the o-nitrobenzyl photo-protecting group is installed on the tyrosine derivatives 2-fluorotyrosine, 3-fluorotyrosine, and 2,6-difluorotyrosine. The directed evolution of an orthogonal amber-tRNA synthetase, specific for these unnatural amino acids, is performed, providing the translational machinery for site-specific incorporation of these compounds. Following expression of protein with the protected tyrosine derivatives, protein exposed to ultraviolet irradiation subsequently loses the protecting group affording the site-specific incorporation of fluorinated tyrosine. Fluorinated tyrosines are introduced to the critical trysoine residue in the chromophore of super-folder green fluorescent protein to determine how the altered pKa affects its fluorescent properties.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.