Synthesis, characterization and X-ray crystal structures of dinuclear nickel(II) complexes of a novel potentially hexadentate but functionally tetradentate binucleating ligand

A binucleating potentially hexadentate chelating agent containing oxygen, nitrogen and sulfur as potential donor atoms (H2ONNO) has been synthesized by condensing alpha,alpha-xylenebis(N-methyldithiocarbazate) with 2,4-pentanedione. An X-ray crystallographic structure determination shows that the Schiff base remains in its ketoimine tautomeric form with the protons attached to the imine nitrogen atoms. The reaction of the Schiff base with nickel(II) acetate in a 1:1 stoichiometry leads to the formation of a dinuclear nickel(II) complex [Ni(ONNO)](2) (ONNO2- = dianionic form of the Schiff base) containing N,O-chelated tetradentate ligands, the sulfur donors remaining uncoordinated. A single crystal X-ray structure determination of this dimer reveals that each ligand binds two low spin nickel(II) ions, bridged by a xylyl group. The nickel(II) atoms adopt a distorted square-planar geometry in a trans-N2O2 donor environment. Reaction of the Schiff base with nickel(II) acetate in the presence of excess pyridine leads to the formation of a similar dinuclear complex, [Ni(ONNO)(py)](2), but in this case comprises five coordinate high-spin Ni(II) ions with pyridine ligands occupying the axial coordination sites as revealed by X-ray crystallographic analysis. (c) 2005 Published by Elsevier B.V.

The structural and electrochemical consequences of hydrogenating Copper N2S2 Schiff base macrocycles

A series of cis and trans tetradentate copper macrocyclic complexes, of ring size fourteen - sixteen, which employ amine and thioether donor groups are reported. Apart from 5,6,15,16-bisbenzo-8,13-diaza-1,4-dithia-cyclohexadecane copper(I) (cis-[Cu(H4NbuSen)]+) all of the complexes are obtained in the copper(II) form. Crystallographic analysis shows that the copper(II) complexes all adopt a distorted planar geometry around the copper. In contrast, cis-[Cu(H4NbuSen)]+ is found to adopt a distorted tetrahedral geometry. The complexes were subjected to electrochemical analysis in water and acetonitrile. The effect of the solvent, positions of the donor atoms (cis/trans) on E1/2 is discussed as is the comparison of the electrochemical behaviour of these complexes with their parent Schiff base macrocycles.

June Sutor and the C-H…O hydrogen bonding controversy

In 1962, D. June Sutor published the first crystallographic analysis of C–H…O hydrogen bonding based on a selection of structures then known. Her follow-up paper the next year cited more structures and provided more details, but her ideas met with formidable opposition. This review begins by describing knowledge of C-H…O hydrogen bonding available at the time from physico-chemical and spectroscopic studies. By comparison of structures cited by Sutor with modern redeterminations, the soundness of her basic data set is assessed. The plausibility of the counter-arguments against her is evaluated. Finally, her biographical details are presented along with consideration of factors that might have impeded the acceptance of her work. © 2012 Taylor & Francis.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Carbon-carbon bond formation methods for the preparation of shape-persistent architectures

Carbon-rich, conjugated organic scaffolding is a popular basis for functional materials, especially for electronic and photonic applications. However, synthetic methods for generating these types of materials lack diversity and, in many cases, efficiency; the insistence of investigators focusing on the properties of the end product, rather than the process in which it was created, has led to the current state of the relatively homogeneous synthetic chemistry of functional organic materials. Because of this, there is plenty of room for improvement at the most basic level. Problems endemic to the preparation of carbon-rich scaffolding can, in many cases, be solved with modern advances in synthetic methodology. We seek to apply this synthesis-focused paradigm to solve problems in the preparation of carbon-rich scaffolds. Herein, the development and utilization of three methodologies: iridium-catalyzed arene C-H borylation; zinc- mediated alkynylations; and Lewis acid promoted Mo nitride-alkyne metathesis, are presented as improvements for the preparation of carbon-rich architectures. In addition, X-ray crystallographic analysis of two classes of compounds are presented. First, an analysis of carbazole-containing arylene ethynylene macrocycles showcases the significance of alkyl chain identity on solid-state morphology. Second, a class of rigid zwitterionic metal-organic compounds display an unusual propensity to crystallize in the absence of inversion symmetry. Hirshfeld surface analysis of these crystalline materials demonstrates that subtle intermolecular interactions are responsible for the overall packing motifs in this class of compounds.

Obtenção e caracterização de dióxido de estanho nanoestruturado pelo método de síntese contínua por combustão em solução

Continuous Synthesis by Solution Combustion was employed in this work aiming to obtain tin dioxide nanostructured. Basically, a precursor solution is prepared and then be atomized and sprayed into the flame, where its combustion occurs, leading to the formation of particles. This is a recent technique that shows an enormous potential in oxides deposition, mainly by the low cost of equipment and precursors employed. The tin dioxide (SnO2) nanostructured has been widely used in various applications, especially as gas sensors and varistors. In the case of sensors based on semiconducting ceramics, where surface reactions are responsible for the detection of gases, the importance of surface area and particle size is even greater. The preference for a nanostructured material is based on its significant increase in surface area compared to conventional microcrystalline powders and small particle size, which may benefit certain properties such as high electrical conductivity, high thermal stability, mechanical and chemical. In this work, were employed as precursor solution tin chloride dehydrate diluted in anhydrous ethyl alcohol. Were utilized molar ratio chloride/solvent of 0,75 with the purpose of investigate its influence in the microstructure of produced powder. The solution precursor flux was 3 mL/min. Analysis with X-ray diffraction appointed that a solution precursor with molar ratio chloride/solvent of 0,75 leads to crystalline powder with single phase and all peaks are attributed to phase SnO2. Parameters as distance from the flame with atomizer distance from the capture system with the pilot, molar ratio and solution flux doesn t affect the presence of tin dioxide in the produced powder. In the characterization of the obtained powder techniques were used as thermogravimetric (TGA) and thermodiferential analysis (DTA), particle size by laser diffraction (GDL), crystallographic analysis by X-ray diffraction (XRD), morphology by scanning electron microscopy (SEM), transmission electron microscopy (TEM), specific surface area (BET) and electrical conductivity analysis. The techniques used revealed that the SnO2 exhibits behavior of a semiconductor material, and a potentially promising material for application as varistor and sensor systems for gas

Analysis of crystallographic slip and grain boundary sliding in a Ti-45Al-2Nb-2Mn (at%)-0.8 vol%TiB2 alloy by high temperature in situ mechanical testing

This work aims to contribute to a further understanding of the fundamentals of crystallographic slip and grain boundary sliding in the γ-TiAl Ti–45Al–2Nb–2Mn (at%)–0.8 vol%TiB2 intermetallic alloy, by means of in situ high-temperature tensile testing combined with electron backscatter diffraction (EBSD). Several microstructures, containing different fractions and sizes of lamellar colonies and equiaxed γ-grains, were fabricated by either centrifugal casting or powder metallurgy, followed by heat treatment at 1300 °C and furnace cooling. in situ tensile and tensile-creep experiments were performed in a scanning electron microscope (SEM) at temperatures ranging from 580 °C to 700 °C. EBSD was carried out in selected regions before and after straining. Our results suggest that, during constant strain rate tests, true twin γ/γ interfaces are the weakest barriers to dislocations and, thus, that the relevant length scale might be influenced by the distance between non-true twin boundaries. Under creep conditions both grain/colony boundary sliding (G/CBS) and crystallographic slip are observed to contribute to deformation. The incidence of boundary sliding is particularly high in γ grains of duplex microstructures. The slip activity during creep deformation in different microstructures was evaluated by trace analysis. Special emphasis was placed in distinguishing the compliance of different slip events with the Schmid law with respect to the applied stress.

Direct structure analysis in protein electron crystallography: crystallographic phases for halorhodopsin to 6-A resolution.

The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.

A crystallographic snapshot of tyrosine trans-phosphorylation in action

Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

Hybrid peptide hairpins containing alpha- and omega-amino acids: Conformational analysis of decapeptides with unsubstituted beta-, gamma-, and delta-residues at positions 3 and 8

The effects of inserting unsubstituted omega-amino acids into the strand segments of model beta-hairpin peptides was investigated by using four synthetic decapeptides, Boc-Lcu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val- OMe: pepticle 1 (Xxx=Gly), pepticle 2 (Xxx=beta Gly=beta hGly=homoglycine, beta-glycine), pepticle 3 (Xxx=gamma Abu=gamma-aminobutyric acid), pepticle 4 (Xxx= delta Ava=delta-aminovaleric acid). H-1 NMR studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for P-hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a beta-hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the gamma Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II' beta-turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 rim, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

Crystallographic Evolution, Dielectric, and Piezoelectric Properties of Bi4Ti3O12:W/Cr Ceramics

W/Cr codoped Bi4Ti3O12 ceramics, Bi4Ti3-xWxO12+x+0.2 wt%Cr2O3 (BITWC, x=0-0.15), were prepared using a solid-state reaction method. The crystallographic evolution and phase analysis were distinctly determined focusing on the X-ray diffraction peak changes in (020)/(200) and (220)/(1115) diffraction planes, by which the lattice parameters, a, b, and c can be refined. The thermal variations of permittivity, dielectric loss (tan delta), impedance, and electrical conductivity properties were characterized. A decrease in the values of Curie temperature from 675 degrees to 640 degrees C and an increase in the values of the dielectric constant due to an increase of W6+/Cr3+ content were observed. The highest piezoelectric constant, d(33) of 22 pC/N, was achieved with the composition of Bi4Ti2.975W0.025O12.025+0.2 wt% Cr2O3. Also, this composition had a lower electrical conductivity than the other investigated compositions.

Structural studies of analgesics and their interactions. XII. Structure and interactions of anti-inflammatory fenamates. A concerted crystallographic and theoretical conformational study

A theoretical conformational analysis of fenamates, which are N-arylated derivatives of anthranilic acid or 2-aminonicotinic acid with different substituents on the aryl (phenyl) group, is reported. The analysis of these analgesics, which are believed to act through the inhibition of prostaglandin biosynthesis, was carried out using semi-empirical potential functions. The results and available crystallographic observations have been critically examined in terms of their relevance to drug action. Crystallographic studies of these drugs and their complexes have revealed that the fenamate molecules share a striking invariant feature, namely, the sixmembered ring bearing the carboxyl group is coplanar with the carboxyl group and the bridging imino group,the coplanarity being stabilized by resonance interactions and an internal hydrogen bond between the imino and carboxyl groups. The results of the theoretical analysis provide a conformational rationale for the observed invariant coplanarity. The second sixmembered ring, which provides hydrophobicity in a substantial part of the molecule, has limited conformational flexibility in meclofenamic, mefenamic and flufenamic acids. Comparison of the conformational energy maps of these acids shows that they could all assume the same conformation when bound to the relevant enzyme. The present study provides a structural explanation for the difference in the activity of niflumic acid, which can assume a conformation in which the whole molecule is nearly planar. The main role of the carboxyl group appears to be to provide a site for intermolecular interactions in addition to helping in stabilizing the invariant coplanar feature and providing hydrophilicity at one end of the molecule. The fenamates thus provide a good example of conformation- dependent molecular asymmetry.

Crystallographic identification of a ‘stepped-cubane’ structure for the Cu4O4 core in [Cu4L2(bipy)4(µ3-OH)2][ClO4]4(HL = 5-hydroxy-6-methylpyridine-,4-dimethanol, bipy = 2,2′-bipyridine)

The structure of [Cu4L2(bipy)4(µ3-OH)2][ClO4]4 containing a Vitamin B6 ligand, pyridoxine (5-hydroxy-6-methylpyridine-3,4-dimethanol, HL), and 2,2′-bipyridine (bipy) has been determined by single-crystal X-ray analysis. This is the first report on a copper(II) complex having a ‘stepped-cubane’ structure. The compound crystallizes in the triclinic space group P[1 with combining macron](Z= 1) with a= 11.015(3), b= 11.902(1), c= 13.142(2)Å, α= 105.07(1), β= 102.22(1) and γ= 99.12(1)°; R= 0.054). The co-ordination geometry around each copper is trigonally distorted square pyramidal. Two of the basal sites are occupied by bipyridyl nitrogens in a bidentate fashion. The remaining basal positions for Cu(1) are filled by a phenolic oxygen and a 4-hydroxymethyl oxygen of the L moiety, whereas for Cu(2) they are occupied by two µ3-OH oxygens. The axial sites are occupied by a µ3-OH oxygen and the 4-hydroxymethyl oxygen of the same pyridoxine for Cu(1) and Cu(2), respectively. Both the bridging nature of the 4-hydroxymethyl oxygen of the L moiety and the unsymmetrical bridging nature of the µ3-OH groups with axial–equatorial bridging are novel features. The structure is discussed in relation to stepped-cubane structures reported in the literature. A comparative study is also made with µ3-hydroxo-bridged copper(II) complexes. Both the plasticity effect of CuII and the stacking interactions between the various rings appear to be important in stabilizing this unusual structure.

Systematic crystallographic investigation of hydrogen-bonded networks involving monohydrogen tartrate-amine complexes: Potential materials for nonlinear optics

Crystal structures of six binary salts involving aromatic amines as cations and hydrogen tartrates as anions are presented. The materials are 2,6-xylidinium-L-monohydrogen tartrate monohydrate, C12H18O6.5N, P22(1)2(1), a = 7.283(2) Angstrom, b = 17.030(2) Angstrom, c = 22.196(2) Angstrom, Z = 8; 2,6-xylidinium-D-dibenzoyl monohydrogen tartrate, C26H25O8N, P2(1), a = 7.906(1) Angstrom, b = 24.757(1) Angstrom, c = 13.166(1) Angstrom, beta = 105.01(1)degrees, Z = 4; 2,3-xylidinium-D-dibenzoyl monohydrogen tartrate monohydrate, C26H26O8.5N, P2(1), a = 7.837(1) Angstrom, b = 24.488(1) Angstrom, c = 13.763(1) Angstrom, beta = 105.69(1)degrees, Z = 4; 2-toluidinium-D-dibenzoyl monohydrogen tartrate, C25H23O8N, P2(1)2(1)2(1), a = 13.553(2) Angstrom, b = 15.869(3) Angstrom, c = 22.123(2) Angstrom, Z = 8; 3-toluidinium-D-dibenzoyl monohydrogen tartrate (1:1), C25H23O8N, P1, a = 7.916(3) Angstrom, b = 11.467(6) Angstrom, c = 14.203(8) Angstrom, alpha = 96.44(4)degrees, beta = 98.20(5)degrees, = 110.55(5)degrees, Z = 2; 3-toluidinium-D-dibenzoyl tartrate dihydrate (1:2), C32H36O10N, P1, a = 7.828(3) Angstrom, b = 8.233(1) Angstrom, c = 24.888(8) Angstrom, alpha = 93.98 degrees, beta = 94.58(3)degrees, = 89.99(2)degrees, Z = 2. An analysis of the hydrogen-bonding schemes in terms of crystal packing, stoichiometric variations, and substitutional variations in these materials provides insights to design hydrogen-bonded networks directed toward the engineering of crystalline nonlinear optical materials.

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the gamma-phosphate of ATP to propionate during L-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate approximate to butyrate), nucleotides (ATP approximate to GTP > CTP approximate to TTP; dATP > dGTP > dCTP) and metal ions (Mg2+ approximate to Mn2+ > Co2+). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, alpha-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the gamma-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer. (C) 2013 Elsevier B.V. All rights reserved.