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

Structural characterization of the major flavonoid glycosides from Arabidopsis thaliana seeds

International audienceInformation gains from the seed of the model plant Arabidopsis thaliana( Brassicaceae) have greatly contributed to a better understanding of flavonoid synthesis and may be used for crop improvement. However, exhaustive identification of the flavonoid accumulated in Arabidopsis seed was still lacking. Complementary investigations of seed flavonoids by LC-ESI-MS, LC-ESI-MS-MS, and NMR spectroscopy in Arabidopsis led to full characterization of monoglycosides, namely, quercetin 3-O-alpha-rhamnopyranoside and kaempferol 3-O-alpha-rhamnopyranoside, and diglycosides, namely, quercetin and kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and quercetin and kaempferol 3,7-di-O-alpha-rhamnopyranoside. Interestingly, the tt7 mutant that lacks flavonoid-3'-hydroxylase and consequently accumulates only kaempferol derivatives was shown to contain three additional products, kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-alpha-rhamnopyranoside-7-O-beta-glucopyranoside, and the triglycoside kaempferol 3-O-beta-[alpha-rhamnopyranosyl(1 -> 2)-glucopyranoside]-7-O-alpha-rhamnopyranoside. Taken together these results allow a scheme for flavonol glycosylation to be proposed

Isotopic Double-Labeling of Two Honeybee Odorant-Binding Proteins Secreted by the Methylotrophic Yeast Pichia pastoris

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Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross- arrangement rich in-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP ( 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully recorded an unprecedented enhancement factor of ε~50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε~50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature

Kingianic Acids A–G, Endiandric Acid Analogues from Endiandra kingiana

A phytochemical investigation of the methanolic extract of the bark of Endiandra kingiana led to the isolation of seven new tetracyclic endiandric acid analogues, kingianic acids A–G (1–7), together with endiandric acid M (8), tsangibeilin B (9) and endiandric acid (10). Their structures were determined by 1D- and 2D-NMR analysis in combination with HRMS experiments. The structure of compounds 9 and 10 were confirmed by single-crystal X-ray diffraction analysis. These compounds were screened for Bcl-xL and Mcl-1 binding affinities and cytotoxic activity on various cancer cell lines. Compound 5 showed moderate cytotoxic activity against human colorectal adeno-carcinoma (HT-29) and lung adenocarcinoma epithelial (A549) cell lines, with IC50 values in the range 15–17 µM, and compounds 3, 6 and 9 exhibited weak binding affinity for the anti-apoptotic protein Mcl-1

Efficient high-field MAS-DNP NMR reveals hidden side chains in amyloid fibrils

It has been widely observed in bioNMR experiments that many biological molecules contain flexible parts or side chains that do not yield easily observable NMR signals in room-temperature experiments. The reasons for the missing peaks could be because the flexible regions might exhibit unfavorable dynamics that interfere with NMR experiments, which result in low NMR intensity below the noise floor. To circumvent this issue, we exploit a hyperpolarization technique known as dynamic nuclear polarization (DNP), which is usually performed at low temperatures for optimal performances. We have also compared 1H enhancements for amyloid fibrils doped with the SNAPol-1 and M-Tinypol radicals, and the 1H DNP spectrum demonstrates a much higher ε~30 for the SNAPol-1 radical than ε~10 for the TinyPol-doped sample. By combining the sensitivity gain bestowed by efficient DNP polarizing agent (SNAPol-1), the freezing of local motions at cryogenic temperature (~ 100 K), and high NMR resolution at high magnetic field (18.8 T), we have successfully recorded an unprecedented enhancement factor of ~50 on amyloid fibrils (NWD2) in magic-angle spinning (MAS) DNP experiments at a high magnetic field of 18.8 T. Moreover, multidimensional MAS NMR experiments have revealed NMR signals of flexible side chains that were previously inaccessible at conventional room-temperature experiments. We also demonstrate that sensitivity-enhanced 2D 15N-13C correlation experiments can be achieved in ~ 2 hours. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains otherwise hidden at room temperature