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In JoVE (1)
Other Publications (21)
- Bioorganic & Medicinal Chemistry
- Journal of Agricultural and Food Chemistry
- Bioorganic & Medicinal Chemistry
- Bioorganic & Medicinal Chemistry
- Bioorganic & Medicinal Chemistry
- Chemical Research in Toxicology
- Journal of Agricultural and Food Chemistry
- Journal of Agricultural and Food Chemistry
- The Plant Journal : for Cell and Molecular Biology
- Biochimica Et Biophysica Acta
- Journal of Agricultural and Food Chemistry
- Solid State Nuclear Magnetic Resonance
- The Journal of Physical Chemistry. C, Nanomaterials and Interfaces
Articles by Ruth E. Stark in JoVE
Isolation and Biophysical Study of Fruit Cuticles
Subhasish Chatterjee1, Sayantani Sarkar1, Julia Oktawiec1, Zhantong Mao1, Olivia Niitsoo2, Ruth E. Stark1
1Department of Chemistry, City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies, 2Department of Chemical Engineering, City College of New York
Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.
Other articles by Ruth E. Stark on PubMed
Titration and Exchange Studies of Liver Fatty Acid-binding Protein with 13C-labeled Long-chain Fatty Acids
Biochemistry. Apr, 2002 | Pubmed ID: 11969406
Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.
Chemical Studies on Antioxidant Mechanism of Tea Catechins: Analysis of Radical Reaction Products of Catechin and Epicatechin with 2,2-diphenyl-1-picrylhydrazyl
Bioorganic & Medicinal Chemistry. Jul, 2002 | Pubmed ID: 11983520
Tea catechins, an important class of polyphenols, have been shown to have wide spectrum of antitumor activity believed to be due mainly to their antioxidative effect. In this study, the radical scavenging behavior of catechins on 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied. Two reaction products of (+)-catechin, and two reaction products of (-)-epicatechin were purified and identified. Their structures were determined on the basis of detailed high-field 1-D and 2-D NMR spectral analysis. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. A possible mechanism of the formation of reaction products is suggested.
Characterization of 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine from Thermal Degradation of Thiamin
Journal of Agricultural and Food Chemistry. Jul, 2002 | Pubmed ID: 12083882
Thiamin hydrochloride was thermally degraded in phosphate buffer (pH 6.5) at 110 degrees C for 2 h. A major decomposition product was isolated by column chromatography and structurally identified by spectrometric techniques ((1)H NMR, (13)C NMR, 2D NMR, and MS) as 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine (MAMP). The possible formation pathway of MAMP was studied using two model systems. It is proposed that MAMP is formed by nucleophilic attack of 2-methyl-3-furanthiol on the thiamin.
Unlocking the Molecular Structure of Fungal Melanin Using 13C Biosynthetic Labeling and Solid-state NMR
Biochemistry. Jul, 2003 | Pubmed ID: 12846559
Melanins are enigmatic pigments found in all biological kingdoms that are associated with a variety of functions, including microbial virulence. Despite being ubiquitous in nature, melanin pigments have long resisted atomic-level structural examination because of their insolubility and amorphous organization. Cryptococcus neoformans is a human pathogenic fungus that melanizes only when provided with exogenous substrate, thus offering a unique system for exploring questions related to melanin structure at the molecular level. We have exploited the requirement for exogenous substrate in melanin synthesis as well as the capabilities of high-resolution solid-state nuclear magnetic resonance (NMR) to establish the predominantly aliphatic composition of l-dopa melanin and to introduce (13)C labels that permit the identification of proximal carbons in the developing biopolymer. By swelling solid melanin samples in organic solvents and using two-dimensional heteronuclear NMR in conjunction with magic-angle spinning, we have identified chemical bonding patterns typical of alkane, alkene, alcohol, ketone, ester, and indole functional groups. These findings demonstrate the feasibility of a novel approach to determining the structure of melanin using metabolic labeling and NMR spectroscopy.
Chemical Studies of the Antioxidant Mechanism of Tea Catechins: Radical Reaction Products of Epicatechin with Peroxyl Radicals
Bioorganic & Medicinal Chemistry. Aug, 2003 | Pubmed ID: 12878131
Tea catechins, an important class of polyphenols, have been shown to have antioxidant activity and are thought to act as antioxidants in biological systems. However, the mechanisms of their antioxidant reactions remain unclear. The objective of this study was to characterize the reaction products of epicatechin with peroxyl radicals generated by thermolysis of the azo initiator azo-bisisobutyrylnitrile (AIBN). Structural elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. Eight reaction products were isolated and identified using high-field 1D and 2D NMR spectral analysis. The observation of these compounds confirmed that the B-ring is the initial site for formation of reaction products in the peroxyl radical oxidant system.
Enzymatic Synthesis of Tea Theaflavin Derivatives and Their Anti-inflammatory and Cytotoxic Activities
Bioorganic & Medicinal Chemistry. Jan, 2004 | Pubmed ID: 14723964
Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.
Bioorganic & Medicinal Chemistry. Jun, 2004 | Pubmed ID: 15142559
Theaflavins and thearubigins are major pigments in black tea, and it is generally accepted that they are produced by oxidation of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the oxidation mechanism of tea polyphenols, especially the formation of thearubigins, a method combining the enzymatic synthesis and LC/ESI-MS/MS analysis was developed to search for new higher molecular weight polymers from black tea. Three new dibenzotropolones, theadibenzotropolone A, B, and C, together with one new tribenzotropolone, theatribenzotropolone A, were formed by the reaction of theaflavins and tea catechins with horseradish peroxidase in the presence of H(2)O(2). The structures of these new benzotropolone derivatives were elucidated on the basis of MS and 2D NMR spectroscopic analyses. The existence of these compounds in black tea was characterized by LC/ESI-MS/MS. Theadibenzotropolone A and B were the first benzotropolone-type trimers of catechins found in the black tea extract. The observation that galloyl ester groups of theaflavins can be oxidized to form di- or tri-benzotropolone skeletons strongly implied that this type of oxidation is an important pathway to extend the molecular size of thearubigins.
Synthesis and Structure Identification of Thiol Conjugates of (-)-epigallocatechin Gallate and Their Urinary Levels in Mice
Chemical Research in Toxicology. Nov, 2005 | Pubmed ID: 16300386
(-)-Epigallocatechin gallate (EGCG), the most abundant and most biologically active compound in tea, has been proposed to have many beneficial health effects. The metabolic fate of EGCG, however, is not well understood. In the present study, we found that EGCG can be oxidized by peroxidase and hydrogen peroxide and then reacted with cysteine or glutathione to form conjugates. The structures of the cysteine and glutathione conjugates of EGCG were identified using 2D NMR and MS. Two thiol conjugates of EGCG (2'-cysteinyl EGCG and 2' '-cysteinyl EGCG) were identified by ESI-LC-MS/MS analysis from the urine samples of mice administered 200 or 400 mg/kg EGCG, i.p. These conjugates were not found in urine samples of mice after receiving EGCG at 50 mg/kg i.p., or 2000 mg/kg i.g., or in human urine following consumption of 3 g of decaffeinated green tea solids (containing 333 mg EGCG). At high doses, EGCG is believed to be oxidized to form EGCG quinone, which can react with glutathione to form the thiol conjugates. These results suggest that detectable amounts of thiol conjugates of EGCG are formed only after rather high doses of EGCG are given to the mice.
Modeling Suberization with Peroxidase-catalyzed Polymerization of Hydroxycinnamic Acids: Cross-coupling and Dimerization Reactions
Phytochemistry. Apr, 2006 | Pubmed ID: 16524605
An anionic potato peroxidase (EC 188.8.131.52, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively.
Magic-angle Spinning NMR Studies of Cell Wall Bound Aromatic-aliphatic Biopolyesters Associated with Strengthening of Intercellular Adhesion in Potato (Solanum Tuberosum L.) Tuber Parenchyma
Biomacromolecules. Mar, 2006 | Pubmed ID: 16529434
Intercellular adhesion strengthening, a phenomenon that compromises the texture and the edible quality of potatoes (Solanum tuberosum L.), has been induced reproducibly by exposure to low-pH acetic acid solutions under tissue culture conditions. The resulting parenchyma tissues have been examined by solid-state nuclear magnetic resonance (NMR) in order to characterize the biopolymer(s) thought to be associated with this syndrome. Cross polarization-magic angle spinning (CPMAS) (13)C NMR has been used to establish the presence of a polyphenol-suberin-like aromatic-aliphatic polyester within an abundant cell wall polysaccharide matrix in potato tubers that exhibit hardening due to strengthened intercellular adhesion. Dipolar dephasing and CP chemical shift anisotropy experiments suggest that the aromatic domain is composed primarily of guaiacyl and sinapyl groups. Two-dimensional wide-line separation experiments show that the biopolymer associated with parenchyma hardening contains rigid polysaccharide cell walls and mobile aliphatic long-chain fatty acids; (1)H spin diffusion experiments show that these flexible aliphatic chains are proximal to both the phenolics and a subpopulation of the cell wall polysaccharides. Finally, high-resolution MAS NMR of parenchyma samples swelled in DMSO in conjunction with two-dimensional through-bond and through-space NMR spectroscopy provides evidence for covalent linkages among the polysaccharide, phenolic, and aliphatic domains of the intercellular adhesion-strengthening biopolymer in potato parenchyma tissue.
Journal of Agricultural and Food Chemistry. Dec, 2006 | Pubmed ID: 17177481
Systematically varied reaction times and concentrations of trifluoroacetic acid (TFA) have been used to remove polysaccharides associated with suberin isolated from potato wound periderm, thereby augmenting spectroscopic determinations of the molecular structure of this protective plant polyester. Treatments with dilute TFA left a residual insoluble material for which both solid-state 13C and 1H NMR spectra displayed significant improvements in resolution without compromising the integrity of the protective plant polyester, whereas higher concentrations of TFA made it possible to achieve controlled hydrolysis of the suberin aliphatic or aromatic domains. Among the isolated fragments were two hydroxyphenyl derivatives reported previously in lignins and a novel aliphatic-aromatic ester trimer that is identified provisionally. Together these protocols help to characterize the carbohydrate types that are bound covalently to the suberin polyester and to identify the interunit covalent linkages among the aliphatic ester, phenolic, and carbohydrate moieties in suberized potato tissue. The strategies described herein may also advance molecular-level investigations of lignocellulosic materials or vegetable tissues that exhibit strengthened intercellular adhesion.
Biochemistry. Nov, 2007 | Pubmed ID: 17927211
Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.
Following Fungal Melanin Biosynthesis with Solid-state NMR: Biopolymer Molecular Structures and Possible Connections to Cell-wall Polysaccharides
Biochemistry. Apr, 2008 | Pubmed ID: 18370403
Melanins serve a variety of protective functions in plants and animals, but in fungi such as Cryptococcus neoformans they are also associated with virulence. A recently developed solid-state nuclear magnetic resonance (NMR) strategy, based on the incorporation of site-specific (13)C-enriched precursors into melanin, followed by spectroscopy of both powdered and solvent-swelled melanin ghosts, was used to provide new molecular-level insights into fungal melanin biosynthesis. The side chain of an l-dopa precursor was shown to cyclize and form a proposed indole structure in C. neoformans melanin, and modification of the aromatic rings revealed possible patterns of polymer chain elongation and cross-linking within the biopolymer. Mannose supplied in the growth medium was retained as a beta-pyranose moiety in the melanin ghosts even after exhaustive degradative and dialysis treatments, suggesting the possibility of tight binding or covalent incorporation of the pigment into the polysaccharide fungal cell walls. In contrast, glucose was scrambled metabolically and incorporated into both polysaccharide cell walls and aliphatic chains present in the melanin ghosts, consistent with metabolic use as a cellular nutrient as well as covalent attachment to the pigment. The prominent aliphatic groups reported previously in several fungal melanins were identified as triglyceride structures that may have one or more sites of chain unsaturation. These results establish that fungal melanin contains chemical components derived from sources other than l-dopa polymerization and suggest that covalent linkages between l-dopa-derived products and polysaccharide components may serve to attach this pigment to cell wall structures.
Journal of Agricultural and Food Chemistry. Nov, 2008 | Pubmed ID: 18828637
Complementary degradative treatments with low-temperature hydrofluoric acid and methanolic potassium hydroxide have been used to investigate the protective biopolymer cutin from Citrus aurantifolia (lime) fruits, augmenting prior enzymatic and chemical strategies to yield a more comprehensive view of its molecular architecture. Analysis of the resulting soluble oligomeric fragments with one- and two-dimensional NMR and MS methods identified a new dimer and three trimeric esters of primary alcohols based on 10,16-dihydroxyhexadecanoic acid and 10-oxo-16-hydroxyhexadecanoic acid units. Whereas only 10-oxo-16-hydroxyhexadecanoic acid units were found in the oligomers from hydrofluoric acid treatments, the dimer and trimer products isolated to date using diverse degradative methods included six of the seven possible stoichiometric ratios of monomer units. A novel glucoside-linked hydroxyfatty acid tetramer was also identified provisionally, suggesting that the cutin biopolymer can be bound covalently to the plant cell wall. Although the current findings suggest that the predominant molecular architecture of this protective polymer in lime fruits involves esters of primary and secondary alcohols based on long-chain hydroxyfatty acids, the possibility of additional cross-linking to enhance structural integrity is underscored by these and related findings of nonstandard cutin molecular architectures.
Phytochemistry. Nov, 2008 | Pubmed ID: 18848340
In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T1(C) spin relaxation measurements, whereas T1rho(H) and T1rho(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T1(C) and T1rho(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.
Cutin Deficiency in the Tomato Fruit Cuticle Consistently Affects Resistance to Microbial Infection and Biomechanical Properties, but Not Transpirational Water Loss
The Plant Journal : for Cell and Molecular Biology. Oct, 2009 | Pubmed ID: 19594708
Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.
Assessing the Size, Stability, and Utility of Isotropically Tumbling Bicelle Systems for Structural Biology
Biochimica Et Biophysica Acta. Mar, 2010 | Pubmed ID: 19914202
Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work, (31)P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl-sn-glycero-3-phosphocholine (DIOHPC), testing their tolerance of variations in commonly used experimental conditions. (1)H-(15)N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.
Isolation and Identification of Triglycerides and Ester Oligomers from Partial Degradation of Potato Suberin
Journal of Agricultural and Food Chemistry. Jan, 2010 | Pubmed ID: 20028122
Suberized cell walls from wound-healing potato tubers ( Solanum tuberosum ) were depolymerized under mild conditions using methanolic potassium hydroxide in order to investigate the chemical linkages present in this protective plant biopolymer. Analysis of the resulting soluble oligomeric fragments with HPLC, 1D and 2D NMR, LC/MS, and MS(n) methods allowed identification of several novel compounds: a family of homologous triglycerides, a family of homologous aliphatic ester trimers, and an ether-linked phenylacetic acid dimer. These findings illustrate the diversity of rigid and flexible molecular linkages present in both poly(aliphatic) and poly(aromatic) domains of potato suberin, and they point toward architectures that may account for its function as a potent hydrophobic barrier to water, thermal equilibration, and microbial pathogens.
Solid State Nuclear Magnetic Resonance. Sep-Oct, 2010 | Pubmed ID: 21036557
A protocol using (207)Pb NMR of solid lead nitrate was developed to determine the temperature of magic-angle spinning (MAS) NMR probes over a range of nominal set temperatures and spinning speeds. Using BioMAS and FastMAS probes with typical sample spinning rates of 8 and 35 kHz, respectively, empirical equations were devised to predict the respective sample temperatures. These procedures provide a straightforward recipe for temperature calibration of any MAS probe.
A Nuclear Magnetic Resonance-based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-binding Proteins
Biochemistry. Mar, 2011 | Pubmed ID: 21226535
Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.
The Journal of Physical Chemistry. C, Nanomaterials and Interfaces. Jan, 2012 | Pubmed ID: 22337562
To efficiently recycle CO(2) to economically viable products such as liquid fuels and carbon nanomaterials, the reactivity of CO(2) is required to be fully understood. We have investigated the reaction of CO(2) with ammonia borane (AB), both molecules being able to function as either an acid or a base, to obtain more insights into the amphoteric activity of CO(2). In the present work, we demonstrate that CO(2) can be converted to graphene oxide (GO) using AB at moderate conditions. The conversion consists of two consecutive steps: CO(2) fixation (CO(2) pressure < 3 MPa and temperature < 100 °C) and graphenization (600-750 °C under 0.1 MPa of N(2)). The first step generates a solid compound that contains methoxy (OCH(3)), formate (HCOO) and aliphatic groups while the second graphenization is the pyrolysis of the solid compound to produce graphene oxide-boron oxide nanocomposites, which have been confirmed by micro-Raman spectroscopy, solid state (13)C and (11)B magic angle spinning-nuclear magnetic resonance (MAS-NMR), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Our observations also show that the mass of solid product in CO(2) fixation process and raw graphene oxide nanocomposites is twice and 1.2 times that of AB initially charged, respectively. The formation of aliphatic groups without using metal-containing compounds at mild conditions is of great interest to the synthesis of various organic products starting from CO(2.).