Browsing by Subject "X-ray crystallography"
Now showing 1 - 17 of 17
Results Per Page
Sort Options
Item Deciphering Lysis and its Regulation in Bacteriophage T4(2012-10-19) Moussa, SamirLike all phages, T4 requires a holin (T) to effect lysis. The lysis event depends on the temporally regulated action of T, which accumulates in the inner membrane (IM) until, at an allele-specific time, it triggers to form a large "hole" in the membrane. Hole formation then releases T4 lysozyme into the periplasm where it degrades the cell wall to elicit cell lysis. Unlike other phages, T4 is unique in exhibiting real-time regulation of lysis based on environmental conditions. Specifically, lysis can be delayed indefinitely in the lysis-inhibited state (LIN), where the normal temporal schedule for holin-triggering is over-ridden. Recently, it was shown that the imposition of LIN was correlated with the interaction of the periplasmic domains (PD) of RI and T. These studies have been extended in this dissertation using genetic, biochemical, and structural techniques to address the molecular mechanism of the RI-T LIN system. First, the PD of RI and an RI-T complex were purified, characterized biophysically, and crystallized to yield the first atomic resolution structures of either a holin or antiholin. The RI PD is mostly alpha-helical that undergoes a conformational change, as revealed by NMR spectroscopy studies, when bound to T. The PD of T is globular with alpha-helical, beta strand, and random coil secondary structures. Additionally, the holin was genetically characterized by mutagenesis techniques, yielding new information on its role in both lysis and LIN. Lysis defective mutants in all three topological domains: cytoplasmic, transmembrane, and periplasmic, were isolated. Analysis of these mutants revealed that both the cytoplasmic and periplasmic domains are important in the oligomerization of T. During LIN, the RI PD binds the PD of T, blocking a holin oligomerization interface. Finally, the signal for the imposition of lysis inhibition has been elucidated using NMR spectroscopy and other in vitro studies. These studies have shown that the RI PD binds DNA. From these studies, new models for lysis and LIN have been constructed. Lysis occurs with the accumulation and oligomerization of T via cytoplasmic and periplasmic domain interactions. LIN is imposed when the ectopically localized DNA of a superinfecting phage interacts with RI, stabilizing it in a conformation competent in inhibiting T oligomerization and leading to lysis inhibition.Item Mechanism of MDA5 Recognition of Short RNA Ligands and Crystal Structure of PepQ(2013-05-16) Watts, Tylan AubreyThe innate immune pathways that stimulate the expression of cytokines and proapoptotic factors in response to infection are triggered by the activation of the cytosolic receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5). Activation of both receptors occurs as a result of binding to RNA. MDA5 only recognizes double stranded forms of RNA, whereas RIG-I is capable of recognizing both single and double stranded RNA. In vivo, MDA5 is known to be stimulated by long (>1 kb) strands of RNA, forming filaments along the phosphate backbone. However, the manner in which MDA5 can recognize the terminal end of its RNA ligand is uncertain. I have examined the mechanism of binding of the MDA5 protein by comparing MDA5 binding to short (<18 bp) blunt RNA, 5? triphosphate RNA, and RNA with a 3? or 5? overhang. It is shown that while the MDA5 protein regulatory domain (RD) is essential for RNA recognition, the MDA5 RD only weakly recognizes short double stranded RNA ligands with overhangs or a 5? triphosphate group. The Cys951 residue was shown to disrupt stability of the MDA5 RD-RNA complex. Binding analyses were performed using a combination of SDS-PAGE, gel filtration analysis, and nondenaturing gel electrophoresis. In addition, structural data was gathered by crystallization of the MDA5 RD-RNA complex using X-ray crystallography. These results help to establish the manner in which MDA5 is regulated predominantly to the binding of long RNA ligands. Also included in this document is structural data on the dimer form of the PepQ protein from E. coli. PepQ is a highly conserved proline peptidase that has a secondary activity of hydrolyzing organophosphorus triesters, toxic compounds found in many pesticides. The PepQ protein was crystallized and analyzed by X-ray diffraction. The dimer interface was clearly defined within the structure and provides insight into how the active dimer forms from the PepQ monomer.Item Mitochondrial tyrosyl-tRNA synthetases : evolving a function beyond translation(2014-08) Lamech, Lilian Tawsein M.; Lambowitz, Alan; Russell, Rick; Johnson, Arlen; Keatinge-Clay, Adrian T; Stevens, Scott; Herrin, David LPezizomycotina mitochondrial tyrosyl-tRNA synthetases (mtTyrRS) are bifunctional, with the ability to splice group I introns in addition to catalyzing aminoacylation. Work done with the Neurospora crassa mtTyrRS (CYT-18 protein) showed that it promotes splicing by binding and stabilizing the conserved catalytically active structure of the intron RNA. To interact with intron RNA, Pezizomycotina mtTyrRSs evolved a new intron-binding surface via structural adaptations on the side of the catalytic domain opposite that which binds tRNA[superscript Tyr]. To examine the variability of these adaptations and intron-binding surface differences between Pezizomycotina mtTyrRSs, I solved the structures of C-terminally truncated C. posadasii and A. nidulans mtTyrRSs. Comparison of these structures to CYT-18 revealed differences in some of the Pezizomycotina specific adaptations that are important for stabilizing key tertiary interactions required for group I intron folding. These studies highlight variations that likely affect intron-RNA binding and potentially splicing and also help define regions for therapeutic intervention. While my and previous studies have provided information on the N-terminal adaptations and intron-binding interactions, little information is available about the Cterminal domain (CTD) interactions. I conducted small angle X-ray scattering (SAXS), binding and splicing assays to further elucidate the domain arrangements of full length CYT-18 and contributions of the CTDs to splicing. My results suggest a model in which free CYT-18 exists in an extended conformation in solution, and upon binding intron RNA, forms a compact structure with both CTDs clamping down onto the RNA. These studies also revealed that the mtTyrRS CTDs have high non-specific binding affinity, which may have facilitated the evolution of the RNA splicing activity in Pezizomycotina mtTyrRSs. Finally, to further investigate the evolution of splicing activity by Pezizomycotina mtTyrRSs, which likely occurred during or after its divergence from Saccharomycotina, I studied bioinformatically reconstructed ancestral mtTyrRSs from the two fungal subphylums. These studies suggest that the common ancestor of the two subphylums may have been capable of non-specifically binding nucleic acid. My research suggests an evolutionary scenario in which an initial non-specific interaction between a self-splicing intron and an ancestral mtTyrRS led to the dependence of the intron on the mtTyrRS for splicing.Item Organometallic Chemistry Supported by the PNP Pincer Framework for Both Early and Late Transition Metals(2012-08-20) Brammell, Christina 1987-Tridentate "pincer" ligands provide a unique balance of stability and reactivity in organometallic chemistry. The development of diarylamido-based PNP pincer ligands has led to many applications in catalysis, including the potential to facilitate unique chemical transformations at transition metal centers. The main objective of this thesis was to explore transition metal chemistry supported by the PNP pincer framework for both early and late transition metals. In Chapter I, the history behind the design and synthesis of pincer complexes is described. The advantages and disadvantages of various pincer ligands are reviewed to show the reasoning behind the synthesis of the PNP pincer framework. Chapter II discusses the synthesis of novel Hf and Ta complexes involving the PNP ligand. Reactions of (PNP)HfCl3 with large alkyl Grignards led to double alkylation and triple alkylation was achieved with methyl Grignard. (PNP)HfMe3 and (PNP)Hf(CH2SiMe3)2Cl displayed remarkably irregular coordination environments about hafnium, in contrast to the approximately octahedral structure of (PNP)HfCl3. (PNP)HfMe3 was found to be thermally stable at 75 degrees C, whereas thermolysis of (PNP)Hf(CH2SiMe3)2Cl under similar conditions led to a mixture of products. The major decomposition product is believed to be a Hf alkylidene complex on the basis of in situ NMR spectroscopic observations (e.g., delta 248.2 ppm in the 13C{1H} NMR spectrum). The reaction of (PNP)TaF4 with an excess of ethyl Grignard led primarily to the double alkylation product, (PNP)Ta(CH2CH3)2F2. Repeating this reaction in the presence of excess ethyl Grignard and dioxane resulted in the formation of an ethylene complex, (PNP)Ta(=CHCH3)(C2H4). In Chapter III, a C-C reductive elimination study is described comparing two pincer ligand scaffolds: Me(PNP) ligand and TH(PNP) ligand. The tied ligand has previously been found to be more sterically demanding than the untied ligand, which has allowed for faster N-C cleavage, faster oxidative addition and a more selective alkyne dimerization catalyst. This study reveals that the tied ligand complex, TH(PNP)Rh(C6H4CF3)(Ph), undergoes slower reductive elimination of p-Ph-C6H4CF3 (< 4% after 7 h at 38 degrees C; t1/2 = 7.7 h at 64 degrees C; t1/2 = 2.13 h at 75 degrees C) than Me(PNP)Rh(C6H4CF3)(Ph) (t1/2 = 15.6 min at 38 degrees C).Item Phosphatases and prolyl-isomerase in the regulation of the C-terminal domain of eukaryotic RNA polymerase II(2012-12) Zhang, Mengmeng; Zhang, Yan Jessie; Robertus, Jon D.; Appling, Dean R.; Siegel, Dionicio R.; Fast, Walter L.In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1.Item Precision x-ray spectroscopy with a three-crystal spectrometer(Texas Tech University, 1968-06) Welch, Herbert EugeneNot availableItem Structural analysis and discovery of lead compounds for the fungal methionine synthase enzyme(2013-12) Ubhi, Devinder Kaur; Robertus, Jon D.Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.Item Structural characterization of synaptotagmin I(2009-02-13) Kerry Fuson; Andres Oberhauser; Xiaodong Cheng; R. Bryan Sutton; Mark Hellmich; Darren BoehningSynaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\r\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\r\nItem Structural studies of the YedU stress protein(2004-11-12) Yonghong Zhao; Robert O. Fox; Vincent J. Hilser; Luis Reuss; Hiram F. Gilbert; David W. BolenThe knowledge of stress proteins is important for understanding stress response, pathology of a broad set of diseases, and the development of therapeutics. The Escherichia coli YedU stress protein, also known as Hsp31, is highly induced upon heat shock. To obtain a better understanding for the possible molecular function of the YedU stress protein, it was expressed, purified, and crystallized. The crystal structure of YedU was determined at 2.2 Å resolution in a multiple isomorphous replacement (MIR) experiment. \r\nYedU monomer has an alpha/beta/alpha sandwich domain and a second smaller domain. Between the sandwich domain and the second smaller domain, there is a putative catalytic triad composed of Cys184, His185, and Asp213. A metal-binding site was identified, where a zinc(II) ion is coordinated by a 2-His-1-carboxylate motif composed of His85, Glu90, and His122. The possible functions of the metal-binding site and the Cys184-His185-Asp213 triad are discussed. \r\nIt was reported that YedU has chaperone activity in vitro. YedU forms dimers in solution and the dimer interface was identified. The molecular surface of the YedU homodimer exhibits a number of solvent-exposed hydrophobic patches that are reminiscent of substrate-binding sites of molecular chaperones. To investigate the role of a central hydrophobic patch in substrate binding, four mutants were made, each replacing a hydrophobic residue with a charged residue. Compared with the wild type YedU protein, the chaperone activity of these mutants was only slightly reduced, suggesting that these residues alone do not play a dominant role in substrate binding at high temperatures.Item Structure based design of a ricin antidote(2012-12) Jasheway, Karl Richard; Robertus, Jon D.; Hackert, Marvin LRicin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This thesis discusses the development of a subset of inhibitors belonging to the pterin family in which amino acids have been utilized as building blocks. Inhibitors in this family have achieved a significant increase in potency, and have provided valuable structural information for further development.Item Studies on cycloaddition reactions of ketenes: further investigations of pseudopericyclic reaction mechanisms(Texas Tech University, 2004-05) Zhou, ChunCycloaddition reactions of ketenes are widely used in organic synthesis. The cycloaddhions of kelene with ethylene, formaldimine and formaldehyde were studied theoretically using B3LYP/6-31G*, MP2/6-31G* and MCSCF/6-31G* methods. A pseudopericyclic mechanism was proposed for the reactions between ketene with ethylene and formaldehyde. A pseudopericyclic transition state was also located for the reaction of kelene with formaldimine even though h has a higher energy barrier than a conrotalory eleclrocyclization transition state, which is a concerted in the gas phase and a stepwise in solvent confirmed by IRC calculations. The cycloadditions of formaldimine with conjugated ketenes were studied theoretically al the B3LYP/6-3IG* level. The concerted and stepwise [4 + 2], [2 + 2] cycloaddition reactions were examined systematically. For the reaction of formaldimine with vinylkelene, the stepwise [2 + 2] and the concerted [4 + 2] are all pericyclic cycloaddhion reactions and have similar energy barriers. For the cycloaddition reactions between formaldimine with imidoylketene and formylketene, the stepwise [4 + 2] pathways are the lowest energy barrier ones. The concerted [4 + 2] and the second step of the stepwise [4 + 2], eleclrocyclization steps are pseudopericyclic with dramatically low barriers. In addition, the choice of formaldimine led the 1, 5- and I, 3-hydrogen shift reaction possible in some zwitterions intermediate, therefore transition stales of them were located. These transition states have low energy barriers and planar geometries. Thus these reactions are best interpreted as pseudopericyclic as well. The least developed conjugated ketenes, imidoylkelenes were studied more experimentally and theoretically. For the first lime, a novel reaction condition was developed to generate a variety of substituted imidoylkelenes. The first biomolecular reactions observed are dimerizations of imidoylkelenes, which were studied experimentally and theoretically. A series of trapping reactions with other reagents were studied as well. In addition, a new method to generate oxokelenes from p-kelo carboxylic acids were investigated and some experimental evidence for the pseudopericyclic reaction mechanism of the thermal chelelropic reactions were found by analyzing the Xray structures of ground states of pyrrolediones, furandiones and their derivatives. Finally, sequential transition states were located in the formation of a semibullvalene and the valley-ridge inflection point was located on its potential energy surface as well.Item Synthesis, crystal structure, and reactivity of a 12-crown-4 sandwich complex of manganese (II)(Texas Tech University, 1980-05) Hampton, Michael DouglasNot availableItem Synthesis, metal complexes, reduction chemistry and antimicrobial applications of a novel bis(imino)acenaphthene (BIAN)-supported N-heterocyclic carbene(2012-12) Butorac, Rachel Renee; Cowley, Alan H; Jones, Richard A; Holliday, Bradley J; Anslyn, Eric V; Brown, Jr., R. MalcolmThe use of N-heterocyclic carbenes (NHCs) as ligands in catalysis is one of the most significant developments in modern catalysis and organometallic chemistry. One way to extend the scope of NHC ligand tuning is by means of annulation of carbocyclic and heterocyclic rings to the NHC backbone. The bis(imino)acenaphthene-supported N-heterocyclic carbene [IPr(BIAN)] has been synthesized and can be regarded as originating from the fusion of a naphthalene ring to an NHC. Several metal complexes of IPr(BIAN), including those incorporating copper(I), silver(I), gold(I), or iridium(I) have been synthesized and characterized, including single-crystal X-ray diffraction studies. The doncity of IPr(BIAN) was investigated using the Tolmen Electronic Parameter (TEP) method. A TEP value of 2042 cm-1 was calculated for the IPr(BIAN) ligand using the Ir(CO)2Cl complex which indicates that IPr(BIAN) is a relatively strong electron donating NHC ligand. The well-behaved redox chemistry of the BIAN ligand class rendered IPr(BIAN) an excellent candidate for exploration of the relationship between ligand charge and carbene donicity. The electrochemical reduction of IPr(BIAN) was studied by cyclic voltammetry (CV) in a THF solution and a reversible reduction wave was detected at - 1.79 V vs SCE. Spectroelectrochemical IR studies were also undertaken to further characterize the nature of the reduced state. IPr(BIAN) was found to be a stronger electron donating ligand in the reduced state in comparison with the neutral state of the ligand. IPr(BIAN) was also chemically reduced using potassium graphite and the resulting radical anion was studied by electron paramagnetic resonance (EPR) techniques. An isotropic EPR signal was observed at a g value of 2.0112. Due to the known antimicrobial activities of silver and gold NHCs, the activities of the silver and gold complexes of IPr(BIAN) and the imidazolium salts of several BIAN ligands were investigated using the minimum inhibitory concentration test. The silver(I) and gold(I) complexes of IPr(BIAN) were found to be moderately active. The most active compounds were found to be the imidazolium salts, with MIC values ranging between < 0.6 μg/mL and 78 μg/mL for the diisopropylphenyl(BIAN) and the mesityl(BIAN) imidazolium chlorides against S. aureas, B. subtilis, E. coli, and P. aeruginosa. The preparation of nanofibers impregnated with IPr(BIAN)AuCl by the process of electrospinning was also explored. The antimicrobial activities of the resulting nanofiber mats were determined on the basis of the inhibition zone test, and a localized antimicrobial activity was observed for the Gram-positive bacteria M. leuteus.Item The synthesis and study of group 13 element-transition metal complexes(Texas Tech University, 1996-12) Johnston, Thomas RichardReactions of indium(I) chloride with low-valent transition metal carbonyl compounds were shown to have only limited utility for producing indium-transition metal bonds. However, the reaction of indium(I) chloride with triruthenium dodecacarbonyl resulted in the synthesis and structural characterization of a new ruthenium carbonyl complex, Ru2(µ-Cl)2(µ-CO)(THF)2(CO)4 (THF = tetrahydrofuran), which contains coordinated tetrah>drofuran. The information gathered from these reactions indicated that indium-transition metal bonds might be formed if indium(I) chloride reacted with low-valent transition metal carbonyls that contain loosely coordinated solvent ligands. These reactions lead to the preparation of two new indiumtransition metal complexes, [(CH3)3NCH2Cl]3[Ru3(µ-InCl3)(µ-InCl2)(µ-CO)(CO)9l(CH3CN)(CH2Cl2)(l)and [(CH3)3NCH2Cl]2[Os(lnCl3)2(CO)4] (2). The crystal structure of 1 showed that the complex contains both the shortest and longest ruthenium-indium bonds yet reported, at 2.592(2) (A) and 2.793(1) (A), respectively. Another interesting feature of this compound is the coordination of InCl3 to two ruthenium atoms. Compound 2 contains the first reported bond between osmium and a Group 13 element other than boron. A reaction of Na2Fe(CO)4 with GaCl3 produced a new iron-gallium compound, Fe[GaCl2(THF)]2(CO)4, which was characterized by X-ray crystallography. The mechanism for this reaction was studied in order to gain a better understanding of the intermediates that are formed. These studies led to the supposition that a solventstabilized Fe(CO)4 fragment is an intermediate species in the reaction. A new method to produce activated indium metal via the disproportionation of indium(I) chloride in tetrahydrofuran was developed. The activated indium metal then reacted with Fe3(CO)i2 to give [Fe(CH3CN)6]2[Fe7ln2(CO)4o](CH3CN)2. The structure of this complex was determined by X-ray crystallography.Item Unveiling the architectures of five bacterial biomolecular machines(2014-08) Fage, Christopher Dane; Keatinge-Clay, Adrian Tristan; Hoffman, David W; Whitman, Christian P; Appling, Dean R; Iverson, Brent L; Hackert, Marvin LNatural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering.Item X-ray line width measurements with a three-crystal spectrometer(Texas Tech University, 1969-05) Welch, Herbert EugeneNot availableItem X-Ray Study of The Transition of Amorphous Nib63s-Pdb17s-Pdb20s Alloy to a Crystalline State(Texas Tech University, 1972-08) Chen, Hua May LinNot Available.