Eric Guittet

Ether linkage between phenolic acids and lignin fractions from wheat straw

Abstract The bonding of the bound-phenolic acids present in three lignin preparations isolated from wheat straw where determined. p -Coumaric acid was mainly ester-linked whereas 35–75% of the recovered ferulic acid was ether-linked to milled straw lignin or enzyme lignin. Ferulic acid ethers accounted for 1.1% dry wt of alkali extracted lignin and might explain the high solubility of Gramineae lignins in soda. Isolated lignins were associated to hemicelluloses, principally arabinoglucuronoxylans. The possible existence of ferulic acid cross-links between lignin and arabinoglucuronoxylans is discussed.

Comparison of Wheat Straw Lignin Preparations - I. Chemical and Spectroscopic Characterizations

Alkali lignin was isolated from wheat straw and compared to two lignin preparations isolated from the same sample by a procedure which allowed to show an heterogeneity of rnonomeric composition between lignin fractions isolated from poplar wood. Using C NMR-, UV-, IRspectroscopy and nitrobenzene oxidation, no significative difference in monomeric composition was found in the case of wheat; difference of condensation degree between these fractions was however suggested by nitrobenzene oxidation. Arabinoxylans, proteins and silica were found in each preparation and a very close association between lignin and polysaccharides is suggested.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases.

Aminoacyl‐tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein–protein or protein–RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl‐prolyl‐tRNA synthetase had a general RNA‐binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled‐coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA‐binding domain appended to eukaryotic aminoacyl‐tRNA synthetases to serve as a cis‐acting tRNA‐binding cofactor.

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.

Photosynthetically 13C-Labelled Poplar Lignins: - 13C NMR Experiments

Poplars were grown from cuttings under air containing CO2 with C at about 20% C, in a growth chamber during three months. C-labelled lignin fractions were isolated and characterized by C NMR spectroscopy. Advantage was taken of the C enrichment to perform C NMR resolution enhancement and editing experiments. Some effects of LiCl, added to the DMSO-d6 Solutions, on the C NMR spectra are discussed. It was also attempted to assign more accurately the polysaccharidic peaks found in the C NMR spectra of lignin fractions in Order to study the lignin-carbohydrate relationships.

Structure of Prokaryotic SECIS mRNA Hairpin and its Interaction with Elongation Factor SelB

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson-Crick base-pairs apart from the GpU are essential for interaction with SelB.

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation.

Heteronuclear dipole-to-dipole cross-relaxation has been applied to exploring intermolecular interactions and intramolecular spatial proximities in a large supramolecular structure comprised of a beta-barrel membrane protein, OmpX, in complex with a polymeric surfactant, amphipol A8-35. The experiments, performed in either the laboratory or the rotating frame, reveal the existence of intermolecular contacts between aromatic amino acids and specific groups of the polymer, in addition to intra-protein dipolar interactions, some of them involving carbonyl carbons. This study opens the perspective of collecting by NMR spectroscopy a new kind of through-space structural information involving aromatic and carbonyl (13)C atoms of large proteins.

The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.