Ruth E. Stark

Structural Evaluation of Phospholipid Bicelles for Solution-State Studies of Membrane-Associated Biomolecules

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.

Tissue- and Cell-Type Specific Transcriptome Profiling of Expanding Tomato Fruit Provides Insights into Metabolic and Regulatory Specialization and Cuticle Formation

This study uses laser capture microdissection coupled with pyrosequencing to characterize the cell- and tissue-type transcriptomes of the pericarp of expanding tomato fruits. This provides new insights into the spatial distribution of expression of structural and regulatory genes associated with many metabolic pathways, and a cuticle lining the inner pericarp surface is described. Tomato (Solanum lycopersicum) is the primary model for the study of fleshy fruits, and research in this species has elucidated many aspects of fruit physiology, development, and metabolism. However, most of these studies have involved homogenization of the fruit pericarp, with its many constituent cell types. Here, we describe the coupling of pyrosequencing technology with laser capture microdissection to characterize the transcriptomes of the five principal tissues of the pericarp from tomato fruits (outer and inner epidermal layers, collenchyma, parenchyma, and vascular tissues) at their maximal growth phase. A total of 20,976 high-quality expressed unigenes were identified, of which more than half were ubiquitous in their expression, while others were cell type specific or showed distinct expression patterns in specific tissues. The data provide new insights into the spatial distribution of many classes of regulatory and structural genes, including those involved in energy metabolism, source-sink relationships, secondary metabolite production, cell wall biology, and cuticle biogenesis. Finally, patterns of similar gene expression between tissues led to the characterization of a cuticle on the inner surface of the pericarp, demonstrating the utility of this approach as a platform for biological discovery.

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss

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.

Chemical studies on antioxidant mechanism of tea catechins: analysis of radical reaction products of catechin and epicatechin with 2,2-Diphenyl-1-picrylhydrazyl

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.

Studies of phospholipid hydration by high-resolution magic-angle spinning nuclear magnetic resonance.

A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

NMR studies of molecular structure in fruit cuticle polyesters

The cuticle of higher plants functions primarily as a protective barrier for the leaves and fruits, controlling microbial attack as well as the diffusion of water and chemicals from the outside environment. Its major chemical constituents are waxes (for waterproofing) and cutin (a structural support polymer). However, the insolubility of cutin has hampered investigations of its covalent structure and domain architecture, which are viewed as essential for the design of crop protection strategies and the development of improved synthetic waterproofing materials. Recently developed strategies designed to meet these investigative challenges include partial depolymerization using enzymatic or chemical reagents and spectroscopic examination of the intact polyesters in a solvent-swelled form. The soluble oligomers from degradative treatments of lime fruit cutin are composed primarily of the expected 10,16-dihydroxyhexadecanoic and 16-hydroxy-10-oxo-hexadecanoic acids; low-temperature HF treatments also reveal sugar units that are covalently attached to the hydroxyfatty acids. Parallel investigations of solvent-swollen cutin using 2D NMR spectroscopy assisted by magic-angle spinning yield well-resolved spectra that permit detailed comparisons to be made among chemical moieties present in the intact biopolymer, the soluble degradation products, and the unreacted solid residue.

The Influence of Water on the Nanomechanical Behavior of the Plant Biopolyester Cutin as Studied by AFM and Solid-State NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticles rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutins elastic properties.

13C chemical shift and 13C-15N dipolar tensors for the peptide bond: [1-13C]glycyl[15N]glycine·HCl·H2O

Abstract The 13 C chemical shift and 13 C- 15 N dipolar tensors have been determined for single crystals of the dipeptide [1- 13 C]glycyl[ 15 N]glycine·HCl·H 2 O. The 13 C chemical shift tensor is axially asymmetric with σ 11 , σ 22 , and σ 33 values of −115.6, −48.6, and 40.6 ppm, respectively, from benzene. The σ 22 component lies approximately along the carbonyl bond and the most shielded component, σ 33 , is perpendicular to the plane defined by the amide oxygen, carbon, and nitrogen. Dipolar splittings in NMR spectra of the doubly labeled material also allow determination of a C-N bond length, yielding a value of 1.39 A which is in good agreement with previous X-ray crystallographic studies.

Chemical studies of the antioxidant mechanism of tea catechins: radical reaction products of epicatechin with peroxyl radicals

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.

Following Fungal Melanin Biosynthesis with Solid-State NMR : Biopolymer Molecular Structures and Possible Connections to Cell-Wall Polysaccharides

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.