Molecular aspects of the interaction between Mason-Pfizer monkey virus matrix protein and artificial phospholipid membrane.

The Mason-Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N-terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid-membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason-Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2 ) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P2 molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P2 molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717-1727. © 2016 Wiley Periodicals, Inc.

Tomentomimulol and mimulone B: two new C-geranylated flavonoids from Paulownia tomentosa fruits.

Two new discovered C-geranylated flavonoids tomentomimulol (1) and mimulone B (2) were isolated from the methanol extract of Paulownia tomentosa (Thunb). Steud. (Paulowniaceae) fruits by exhaustive chromatographic separation together with one known compound tanariflavanone D (3). The identification of compounds and structure elucidation was carried out using 1D and 2D NMR experiments, as well as mass spectroscopy, ultra-violet, infra red and CD experiments.

Towards regioselective synthesis of oligosaccharides by use of alpha-glucosidases with different substrate specificity.

alpha-Glucosidase from two microbial sources, Bacillus stearothermophilus and Brewer's yeast, has been used to catalyze transglycosylation reactions and a comparative study was carried out to determine the regioselectivity of this reaction. Bacterial alpha-glucosidase exhibited higher transfer activity with maltose and was able to synthesize tri- and tetrasaccharides in high yield (27%). In the case of yeast enzyme, only trisaccharides were synthesized in lower yield. Structure analysis of transglycosylation products by means of GC-MS and NMR spectroscopy revealed a correlation between the hydrolytic substrate specificity and the regioselectivity of transglycosylation reaction. Higher substrate specificity of bacterial enzyme, however, influenced its transglucosylation activity toward other saccharide acceptors.

The hairpin stack fold, a novel protein architecture for a new family of protein growth factors.

The granulin/epithelin protein motif has an unusual structure consisting of a parallel stack of beta-hairpins stapled together by six disulphide bonds. The new structure also contains a folding subdomain shared by small toxins, protease inhibitors as well as the EGF-like protein modules.

Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin.

The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes.

Gradient-enhanced TOCSY experiments with improved sensitivity and solvent suppression.

Gradient-enhanced versions of the homonuclear TOCSY experiment are described, with solvent suppression and sensitivity superior to that of a conventional TOCSY experiment. The pulse sequences are constructed by appending a WATERGATE module to a z-filtered TOCSY experiment. Pulsed-field gradients and appropriately phased selective rf pulses are used to maintain precise control of the water magnetization vector. Problems associated with radiation damping and spin-locking of the water magnetization are thus alleviated. The water magnetization is returned to equilibrium prior to each acquisition, which improves water suppression and minimizes signal losses due to saturation transfer.

Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects.

The EGF-like domains in human thrombomodulin interact with and change the specificity of thrombin from a procoagulant enzyme to an anticoagulant enzyme. Recent experiments identified the minimal thrombin-binding region of thrombomodulin as the most acidic loop of the fifth EGF-like domain with a sequence of E408CPEGYILDDGFI420CTDIDE. High-resolution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys1(409)Pro2Glu3Gly4Tyr5Ile6- Leu7Asp8Asp9Gly10Phe11Cys12Thr13Asp14Ile15Asp16Glu17(426), with its target coagulation protein, thrombin. The disulfide-bonded peptide was found to be structured only upon binding, while neither the linear nor the cyclized peptide exhibited any structural preference free in solution. The thrombin-bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear Overhauser effects (transferred NOEs) and by distance geometry and Monte Carlo calculations. The thrombin-bound cyclic peptide assumes an overall conformation similar to those observed in the free but intact EGF molecules. There is a type II beta-turn involving residues Pro2-Tyr5, followed by an optimized antiparallel beta-sheet involving residues Gly4-Asp8 and residues Phe11-Ile15. The thrombomodulin peptide provides a potential thrombin-binding surface between residues Tyr5 and Phe11, which are brought close by a chain reversal within the central beta-sheet. Comparison of the thrombin-bound structure of the EGF-like subdomain with other thrombin-peptide complexes revealed that a common thrombin-binding surface can be organized by different secondary structure elements with entirely different peptide sequences. The thrombin-bound structure of the thrombomodulin peptide may serve as a basis to understand the regulatory functions of thrombomodulin and as a guide for the design of specific inhibitors for thrombin.

Metabolic pathways of 1-butyl [3-13C]acrylate. Identification of urinary metabolites in rat using nuclear magnetic resonance and mass spectroscopy.

1-Butylacrylate, an industrial monomer, is rapidly metabolized by carboxylesterase-catalyzed hydrolysis to acrylic acid and 1-butanol. Acrylic acid enters the intermediary metabolism and is efficiently degraded to carbon dioxide as the metabolic end product. To obtain a virtually complete metabolic pattern, rats were dosed by a single intraperitoneal dose of 1 mmol/kg 1-butyl [3-13C]acrylate. The urine was then analyzed by a one-dimensional 1H-detected and two-dimensional 1H-13C shift-correlated heteronuclear multiple-quantum NMR experiment. In this experiment, three urinary metabolites, namely, 3-hydroxypropanoic acid, N-acetyl-S-(2-carboxyethyl)cysteine, and N-acetyl-S-(2-carboxyethyl)cysteine sulfoxide, were identified comparing their 1H and 13C chemical shifts with those of authentic standards. In another experiment, to enhance minor metabolic pathways, rats were dosed with 0.25 mmol/kg of a carboxylesterase inhibitor, tri-o-tolyl phosphate, prior to 0.5 mmol/kg butyl [3-13C]acrylate. Under these conditions, N-acetyl-S-(2-carboxyethyl)cysteine, N-acetyl-S-[2-(butoxycarbonyl)-ethyl]cysteine, and N-acetyl-S-(2-carboxyethyl)cysteine sulfoxide were found in urine. No metabolites which would arise from a possible metabolic activation of 1-butyl acrylate to 1-butyl oxiranecarboxylate and its subsequent hydrolysis or glutathione conjugation were found. It is estimated that any metabolite amounting to more than 1% of the dose should be detected under these conditions. To study the routes by which BA enters the intermediary metabolism, incorporation of the label into urinary carboxylic acids was followed by GC/MS. Significant enrichment was found in 3-hydroxypropanoic acid and citric and isocitric acid but not in lactic acid.(ABSTRACT TRUNCATED AT 250 WORDS)