Chemistry & Biochemistry
Permanent URI for this communityhttp://hdl.handle.net/1903/11812
Browse
172 results
Search Results
Item A Matrix Universe as the Origin of Inertia and Momentum and Translocation as a Chemical Reaction(2003-03) Hansen, John NormanIt is proposed that there is a matrix structure of the universe that persists both in the presence and absence of physical objects, and that chemical reactions involve reorganization of electronic orbitals within this matrix structure. The mechanism by which objects translocate within this matrix is similar to the chemical reaction mechanism, and therefore requires moving objects to cross transition-state energy barriers. This involves an input of energy, which is the origin of inertia and momentum. The matrix concept is used to derive equations of motion that conform to Newton’s laws and include terms for inertia and momentum.Item v-RIO1 – an atypical protein kinase from the parasitic nematode Trichostrongylus vitrinus(Springer Nature, 2008-09-22) Hu, Min; LaRonde-LeBlanc, Nicole; Sternberg, Paul W; Gasser, Robin BProtein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1). This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.Item Solvation, Structure and Organization at Liquid Surfaces(2009) Brindza, Michael Ross; Walker, Robert A; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation presents the results of nonlinear spectroscopic studies whose goal is to understand how the asymmetric nature of interfaces and intermolecular interactions give rise to interfacial solvation properties and solvent structure. The first part of this thesis uses resonance enhanced second harmonic generation to examine the polarity and hydrogen bonding opportunities at interfaces formed between hydrophilic silica and both weakly and strongly associating organic liquids. Measuring interfacial electronic spectra of probe molecules that exhibit solvatochromic sensitivity to polarity and hydrogen bonding, we saw that small changes in solvent structure affect interfacial polarity, and strongly associating alcohols solvents create a region of heterogeneous polarity at the interface. Silica appears to donate hydrogen bonds to adsorbates no matter what solvent (protic or aprotic) was chosen. The second part of this dissertation uses another nonlinear spectroscopic technique, vibrational sum frequency generation, to determine the structure and orientation of solvent molecules adsorbed to silica/vapor, silica/liquid, and neat liquid/vapor interfaces. By comparing spectral features appearing under different experimental polarization conditions, we have determined average solvent orientations and degree of organization. Our initial studies of alkanes adsorbed to the silica/vapor interface show that despite strong substrate-adsorbate interactions, molecules at the interface show some degree of long range order and organization. In order to examine how the strength of intermolecular forces between adsorbates and either the substrate or neighboring molecules affect interfacial organization, we measured vibrational spectra of octanol isomers as well as different functional group containing n-alkyl molecules at silica/vapor and silica/liquid interfaces. The octanol studies show that strongly associating molecules form ordered monolayers at the silica/vapor interface, but that strength of lateral interactions is important for preserving that order when the liquid is brought into contact. Branched isomers appeared very disordered at solid/liquid interfaces. Further examining this change in order between solvents at silica/vapor and silica/liquid interfaces using equal length but different functional group containing solvents, we see that the energetics of adsorption and solvation are likely to be responsible for the degree of order both at the solid/vapor surface (adsorption) and solid/liquid interface (both adsorption and solvation).Item SCANNING TUNNELING MICROSCOPY / SPECTROSCOPY STUDIES OF BINARY ORGANIC FILMS(2009) Jin, Wei; Reutt-Robey, Janice E; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Multi-component organic molecular films have seen increasing applications in photovoltaic technologies and other organic electronic applications. These applications have been based upon assumptions regarding film structure and electronic properties. This thesis provides an increased understanding of factors that control structure in binary molecular films and begins to establish structure-electronic property relations. In this thesis, three technologically relevant "donor-acceptor" systems are studied with variable temperature STM/STS: pentacene (Pn):C60, zinc phthalocyanine (ZnPc): C60 and ZnPc: perfluorinated zinc phthalocyanine (F16ZnPc). These three model systems provide a systematic exploration of the impact of molecular shape and molecular band offset on morphology-electronic relations in thin film heterostructures. For Pn:C60, I show how domain size and architecture are controlled by composition and film processing conditions. Sequential deposition of pentacene, followed by C60, yields films that range from nanophase-separated, to co-crystalline phases, to a templated structure. These distinct structures are selectively produced from distinct pentacene phases which are controlled via pentacene coverage. For the ZnPc:C60 system, the shape of ZnPc and the lattice mismatch between ZnPc and C60 are quite different from the Pn:C60 films. Nonetheless, ZnPc:C60 films also yield chemical morphologies that can be similarly controlled from phase separated, to co-crystalline phases, to templated structures. In both of these binary films, I exploit relative differences in the component cohesive energies to control phase selection. In bilayer films of both systems, a common structural element of stress-induced defects is also observed. In ZnPc:F16ZnPc, I explore two components with similar shapes and cohesive energies while retaining molecular band offsets comparable to Pn:C60. In this shape-matched system, a checkerboard ZnPc:F16ZnPc arrangement stabilized by hydrogen bonds readily forms. This supramolecular structure introduces a new hybridization state close to the Fermi Level, yielding electronic properties distinct from the component phases. Through investigations of these three model systems, I have developed an understanding the control of chemical morphology along the donor-acceptor interface and the way this morphology influences electronic transport.Item REVERSIBLE QUINONE METHIDE ALKYLATION OF DNA(2009) Wang, Huan; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alkylation of DNA has been found to cause cancer and also to serve as its treatment. Quinone methides (QMs) are highly electrophilic molecules implicated in numerous metabolism processes. Studies of QM's reversible reaction with nucleophiles of DNA are important to understand the mechanism of its biological activity. Reversible alkylation of QMs can extend their lifetime under aqueous conditions. The repeated capture and release of QM from dA adduct can help QM equivalents escape the irreversible trapping and extend QM's lifetime by 100-fold. This effect of dA saturates at a concentration of about 6 mM. In contrast, dG, dC, and dT do not have the ability to preserve QM under aqueous conditions. Oligonucleotides can also preserve QM equivalents by forming labile intrastrand adducts. An oligonucleotide has now been shown to transfer bisQM to its complementary sequences to form interstrand crosslinking. Non-complementary sequences can not be alkylated by bisQM-oligonucleotide adducts. The nucleotide composition of oligonucleotides affects their ability to transfer QM as well. A G rich sequence showed a strong ability for crosslinking a complementary sequence. However, C rich and A rich sequences did not have such an ability. Excess alkylation of C rich and A rich oligonucleotides relative to that of G rich oligonucleotide may interrupt the hybridization of complementary sequences and suppress the formation of DNA crosslinking. The reversibility of crosslinking by QM within duplex DNA has been demonstrated by a strand displacement system. The reversible QM-DNA bond does not prevent strand displacement and allows bisQM to migrate within a series of changing DNA structures by forming crosslinking. The reactivity of bisQM is preserved beyond 11 days in duplex DNA by forming labile DNA cross-links under aqueous conditions. The migration of QM is found to be under thermodynamic control and bisQM preferentially retain cross-links in the most stable DNA duplexes.Item Visible Light Photorelease of Carboxylate Anions by Mediated Photoinduced Electron Transfer to Pyridinium-based Protecting Groups(2009) Borak, John Brian; Falvey, Daniel E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The use of sensitized photoinduced electron transfer (PET) to trigger release of redox-active photoremovable protecting groups (PRPGs) allows a broad range of chromophores to be selected that absorb in difference wavelength ranges. Mediated electron transfer (MET) is particularly advantageous as sub-stoichiometric amounts of the often costly sensitizer (relative to the amount of protected substrate) can be combined with an excess amount of an inexpensive electron donor. Thus, the sensitizer acts as an electron shuttle between the donor and the protecting group to initiate release. The development of improved MET release systems using visible light as the trigger is the focus of the current work. The N-alkylpicolinium (NAP) group has demonstrated its utility as an aqueous-compatible PET-based PRPG, releasing protected substrates upon one electron reduction. Adaptation of MET PRPG release to visible light absorbing mediators began with employing ketocoumarin dyes that primarily form excited triplet states. These chromophores demonstrated high rates of release of NAP-protected carboxylates using sub-stoichiometric concentrations of mediator. Subsequently, nanomolar concentrations of gold nanoparticles were used to mediate electron transfer to NAP-protected compounds. This system exhibited rapid deprotection with very high release quantum efficiencies. In an effort to use highly stable visible-light-absorbing metal-centered dyes with modest redox properties, the NAP group has been synthetically modified to adjust its reduction potential to more positive values. Photolysis of solutions containing the protected substrate, a large excess of an electron donor, and substoichiometric amounts of the dye tris(bipyridyl)ruthenium(II) released the free carboxylates in high yields while photodegradation of the chromophore was minimal. To demonstrate the utility of the NAP group, a quasi-reversible photorheological fluid has been developed based on the formation and disruption of aqueous micelles. In solutions containing the surfactant cetyltrimethylammonium bromide, visible light photorelease of a carboxylate additive from the NAP-ester derivative induces a 105 increase in solution viscosity due to the formation of an interpenetrating micelle network. Subsequent irradiation of the viscoelastic fluid with UV light induces a cis-trans isomerization within the released carboxylate thereby disrupting the micelle network and decreasing solution viscosity by 102.5.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 A Novel Pre-fluorescent Nitroxide Probe for the Highly Sensitive Determination of Peroxyl and Other Radical Oxidants(2009) Jia, Min; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Peroxyl and other radical oxidants react with stable cyclic nitroxides, such as the piperidinyl and pyrrolidinyl nitroxides to form initially the one electron oxidation product, the oxoammonium cation. For most of the nitroxides studied thus far, the oxoammonium cation can in part be regenerated to the nitroxide through reduction by solution constituents. The reaction mechanisms, however, remain a matter of debate. Further, the highly-sensitive, quantitative determination of peroxyl and other radical oxidants has yet to be achieved, posing a major hurdle to a further understanding of the impact of peroxyl radicals in many biological and environmental processes. A unique, amino-pyrrolidinyl nitroxide, 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a diamagnetic product. When a fluorophore, fluorescamine is covalently linked through the amino group on the nitroxide, the resulting compound (3-apf, or I) has very low fluorescence quantum yield. Upon reaction with peroxyl and other radical oxidants, the quantum yield of the product increases dramatically (~100 fold), and thus 3-ap or 3-apf can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity using a spectrofluorometer, by HPLC analysis with fluorescence detection, or by a combination of both approaches. By changing the [O2]/[nitroxide] ratio, it is shown that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants, such as *NO2 and CO3*-. When decreasing the [O2]/[nitroxide] ratio, the oxidation product decreases, with a concomitant increase of the alkoxylamine product resulting from reaction of 3-ap (3-apf) with carbon centered radicals. Preliminary studies suggest that the reactions of 3-ap and 3-apf with peroxyl radical produce different final products. High resolution mass spectrometry and NMR studies indicate that 3-ap is oxidized to form a cyclic peroxide structure, while 3-apf is oxidized to form a cyclic -NH-O- structure, with this difference resulting possibly from the presence of the fluorescamine moiety in 3-apf. Detection of photochemically produced peroxyl radicals is achieved by employing 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) alone, followed by derivatization with fluorescamine, while detection of thermally-generated peroxyl radicals employs 3-apf. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes by 3-apf and produced photochemically in tap water by 3-ap.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 Catechols as Membrane Anion Transporters(2009) Berezin, Sofya; Davis, Jeffery T.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Document: CATECHOLS AS MEMBRANE ANION TRANSPORTERS Sofya Berezin, Doctor of Philosophy, 2009 Directed By: Professor, Jeffery T. Davis, Department of Chemistry and Biochemistry Synthetic anion transporters have potential as antimicrobials, extractants, sensors, etc. Anionophores may also help us understand how natural systems move ions across hydrophobic barriers. While bacterial siderophores and synthetic analogues use catecholates for Fe3+ uptake, this work reports of catechols facilitating biomembrane transport of anions. We demonstrate that simple bis-catechol III-25 is an anion transporter whose activity depends on catechol's substitution and amphiphilicity. We also describe liposomal assays and devised quantitative description that allows one to study facilitated anion transport. These assays indicate that selectivity of III-25 follows the Hofmeister bias: anions which are easier to dehydrate are made more permeable to the membrane by this bis-catechol. We believe that our description of the ion selectivity and mechanism for III-25 opens an outstanding opportunity for those interested in determining the selectivity and mechanism for other synthetic and natural biomembrane ion transporters. In the beginning of this project we investigated number of simple amides and phenols to evaluate their relative affinity and stoichiometry of interaction with Cl- anion. ESI-MS and 1H NMR analysis showed that a dimer, catechol2*Cl-, was the major complex formed when TBA+Cl- was mixed with excess catechol. Based on this finding we attached two catechols to a TREN scaffold. A hydrophobic alkyl amide groups were linked to TREN's third position. Surprisingly, this simple design led to the active analogs III-23 - III-26. A medium-length, III-25, was the most active compound, indicating that ion transport ability depends on the ability to partition into the biomembrane. Finally, we noticed that the experimentally observed weak dependence of the transport rates on the anion's hydration energy, namely, kAnion decreasing in the order ClO4- > I- > NO3- > Br- > Cl-, is also seen for some of Nature's anion transporters. Thus, anion permeation into the CFTR chloride channel shows a similar trend. We also observed a nonlinear dependence of kAnion on the concentration of bis-catechol. These findings led us to believe that self-association of III-25 provides transient pores that allow permeation without requiring complete dehydration of the inorganic anions. Future efforts will include incorporating selectivity filters into these bis-catechols to help overcome the Hofmeister bias.