From meiogynin A to the synthesis of dual inhibitors of Bcl-xL and Mcl-1 anti-apoptotic proteins.

The synthesis of one of the most potent dual inhibitors of the anti-apoptotic proteins Bcl-xL and Mcl-1 is reported. This analogue of a natural sesquiterpenoid dimer meiogynin A was elaborated by a convergent asymmetric synthesis with 36% yield in ten steps.

Two triterpene saponins from Achyranthes bidentata.

Bidentatoside II(1) and chikusetsusaponin V methyl ester (2) are two further triterpene saponins isolated from the roots of Achyranthes bidentata. Chemical and homo and heteronuclear two-dimensional (2D) NMR techniques have led to the structural elucidation of 1 which is a new seco-glycoside of oleanolic acid and the full 1H- and 13C-NMR assignments of 2. These compounds did not show any potentiation of the in vitro cytotoxicity of cisplatin in the HT 29 human colon cancer cell line.

Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris.

Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.

Petersaponins III and IV, triterpenoid saponins from Petersianthus macrocarpus.

Two new triterpenoid saponins, petersaponins III and IV (1 and 2), were isolated from an n-butanol extract of the bark of Petersianthus macrocarpus. They possess 21-O-benzoyl-22-O-acetylbarringtogenol C and 21-O-2-furoxyl-22-O-tigloylbarringtogenol C as the aglycon, respectively. For both 1 and 2, the trisaccharide moiety linked to C-3 of the aglycon consists of D-glucuronic acid, D-xylose, and D-galactose, while a L-rhamnose unit is linked to C-28. The structures of 1 and 2 were elucidated by extensive NMR experiments including (1)H-(1)H (COSY, HOHAHA, NOESY) and (1)H-(13)C (HMQC and HMBC) spectroscopy and by chemical evidence.

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.

Study of amide proton exchange in 15N-enriched cryptogein using pH-dependent off-resonance ROESY-HSQC experiments.

A new pH-dependent off-resonance ROESY-HSQC experiment has been used to characterize the degree of protection of the amide protons of cryptogein, a protein of the elicitin family, against solvent exchange. The study of the pH dependence of solvent-shielded amide protons in this protein reveals that the helices have different levels of stability. Two of the five helices exhibit strong protection of amide hydrogens against exchange with the solvent. By contrast, greater flexibility is observed in the other three helices, particularly in the C-terminal helix. These results provide information on the dynamic features of the protein and are consistent with the RMSD for the backbone atoms of residues involved in helical structures. In addition, the question of the flexibility in a hydrophobic cavity made of conserved residues, which represent a plausible binding site, is addressed by this method.

A study of protein-water exchange through the off-resonance ROESY experiment: application to the DNA-binding domain of AlcR.

In this communication a new NMR experiment for the safe observation and quantification of water-protein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shift-based separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.

Solution structure of a Lewis(x) analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference.

A recent 1H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis(x). Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters (tau(cp)). With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.

Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]-enkephalin in aqueous solution as revealed by the combined use of 17O-NMR and Fourier-transform infrared spectroscopy.

Solvent-induced and temperature-induced 17O chemical shifts of [17O-Gly2, Leu5]-enkephalin and [17O-Gly3, Leu5]-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu5]-enkephalin and [1-13C-Gly2, Leu5]-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 17O-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.

17O NMR and FT-IR study of the ionization state of peptides in aprotic solvents. Application to Leu-enkephalin.

The ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions.

17O-NMR studies of the conformational and dynamic properties of enkephalins in aqueous and organic solutions using selectively labeled analogues.

The synthesis of Leu-enkephalin selectively 17O-enriched in Gly2 and Gly3 is reported. The 17O-nmr chemical shifts of [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in H2O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical and independent of the pH. The 17O chemical shifts of the [17O-Leu5]-enkephalin terminal carboxyl group at pH approximately 1.9 and 5.6 are very different in H2O but very similar in CH3CN/DMSO (4:1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH3CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH approximately 5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (delta delta approximately 30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2----5 hydrogen bonding exists to an appreciable extent. This conclusion is in agreement with the energy of activation for molecular rotation, as determined from T1 measurements, which was found to be almost identical for both [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in CH3CN/DMSO (4:1) mixed solvent.

A crystal molecular conformation of leucine-enkephalin related to the morphine molecule.

Leucine-enkephalin (Try1-Gly2-Gly3-Phe4-Leu5) has been crystallized as a trihydrate from water solution. X-ray diffraction reveals a tightly folded molecular conformation with two fused beta III- (Gly2-Gly3) and beta I- (Gly3-Phe4) turns. The Tyr1 and Phe4 aromatic rings have a close orthogonal arrangement analogous to the tyramine and cyclohexenyl rings in morphine. This suggests that the conformation found in the trihydrate crystal structure could be required for recognition by mu-receptor sites.