Search

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein&ndash, protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

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.

The interactions of fungal guilds have recently been proposed as drivers of organic matter transformation in forest soils. We conducted a pot experiment with Norway spruce seedlings planted in spruce needle litter inoculated with several fungal strains belonging to different ecological guilds (saprotrophic, mycorrhizal, and root endophytic) to assess how the fungi and their interactions affect the transformation of humic substances (HS) and phosphorus (P) in the litter. Several methods for the characterization of P forms and HS were employed, including 31P NMR, UV–Vis and FTIR spectroscopy. Our results show that fungal interactions influence not only the flow of P in decaying (plant) litter but also the transformation of the soil organic matter itself. Pots with saprotrophic Gymnopus androsaceus generally retained more P and prevented the accumulation of phosphonates caused by mycorrhizal Hyaloscypha finlandica, highlighting the strong competitive ability of the former species. The increased mineralization of P caused by G. androsaceus was not observed in the combined treatments, suggesting that other present fungi took up part of the inorganic P. The tested fungi did not affect the amount of HS produced but changed the characteristics of the HS. Mycorrhizal H. finlandica and root endophytic Phialocephala fortinii increased the relative proportion of carboxylic moieties in the HS regardless of the presence or absence of G. androsaceus, probably via the production and incorporation of melanins. The UV–Vis absorbance characteristics of the HS were significantly influenced by fungal interactions. Mycorrhizal H. finlandica and Hebeloma bryogenes retarded humification, as determined by the A4/6 ratio. We attribute the similar shift observed in Serpula himantoides to the partial oxidative degradation of HS. Our study shows that fungal root endophytes can significantly contribute to litter transformation along with mycorrhizal and saprotrophic fungi. The extent and patterns of the transformation seem to be species-dependent in all studied fungal groups.

The composition and structure of humic acids isolated from lignohumate, which is produced by hydrolytic–oxidative conversion of technical lignosulfonates, were characterized by chemical and spectral methods (UV/VIS, FTIR, and 13 C NMR spectroscopy). As comparative samples, humic acids (HA) were isolated also from lignite and organic horizon of mountain spruce forest soil. When compared with other HA studied, the lignohumate humic acids (LHHA) contained relatively few carboxyl groups, whose role is partly fulfilled by sulfonic acid groups. Distinctive 13 C NMR signal of methoxyl group carbons, typical for lignin and related humic substances, was found at the shift of 55.9 ppm. Other alkoxy carbons were present in limited quantity, like the aliphatic carbons. Due to the low content of these carbon types, the LHHA has high aromaticity of 60.6%. Comparison with the natural HA has shown that lignohumate obtained by thermal processing of technical lignosulfonate can be regarded as an industrially produced analog of natural humic substances. Based on the chemical and spectral data evaluation, structural features of lignohumate humic acids were clarified and their hypothetical chemical structure proposed, which described typical “average” properties of the isolated fraction.

The matrix protein (MA) of the Mason-Pfizer monkey virus (M-PMV) plays a key role in the transport and budding of immature retroviral particles from the host cell. Natural N-terminal myristoylation of MA is essential for the targeting of the particles to the plasma membrane and participates in the interaction of MA with membranes phospholipids. The mutation Y28F/Y67F in MA reduces budding and thus causes the accumulation of viral particles under the cytoplasmic membrane. To investigate the impact of Y28F/Y67F mutation on the structure of MA, we prepared this protein in amount and quality suitable for NMR spectroscopy. We report backbone, side-chain and myristoyl residue assignments of the Y28F/Y67F mutant of the M-PMV matrix protein, which will be used to study the interaction with membrane phospholipids and to determine the structure of the mutant matrix protein.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon 13 C and nitrogen 15 N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly 13 C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8 mg of the myristoylated, doubly labeled ( 13 C/ 15 N)M-PMV matrix protein from 1 L of 15 N/ 13 C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U- 13 C]myristic acid.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-d-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.

Specific chemical modification is one of the basic techniques of protein chemistry. Inter alia can be used for detection of surface accessible amino acid residues; this information is of particular importance for studies of the participation of residues in intermolecular interactions of a protein. We achieved an improvement of the technique for arginine, aspartic, and glutamic acid modification using a simple combination of gel permeation and reversed-phase chromatography prior to mass spectrometry analysis. The improved protocol was tested on cytochrome c and M-PMV matrix protein. In both proteins, all accessible arginines and a high number of acidic amino acids were modified. These results indicate that the new protocol can be useful in protein structure analysis, generally.

Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the 1 H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our in vivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C 8 fatty acid chains was monitored by observation of concentration‐dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in 31 P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein–phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a 13 C-filtered/ 13 C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P 2 binding was not strong enough for triggering of the myristoyl‐switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

In our screening, we aimed to detect phosphonates and other forms of organic phosphorus in basidiocarps and vegetative mycelia of six common basidiomycetes. Organic phosphorus-containing compounds were extracted in alkali and analysed using 31 P NMR. Monoesters, diesters, pyrophosphates and polyphosphates detected in high amounts reflected the high metabolic activity in basidiocarps (growth, production of basidiospores). Phosphonates were present in all samples, in concentrations ranging from 14 mg kg −1 of the extracted phosphorus in Boletus badius basidiocarp to 140 mg kg −1 in Amanita muscaria vegetative mycelium. Detection of phosphonates in basidiocarps together with our previous evidence from laboratory experiments support the fungal production of natural phosphonates in forest ecosystems.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason–Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMVPRC7A/C106A). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMVPRC7A/C106A was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Fungal decomposition of and phosphorus transformation from spruce litter needles ( Picea abies ) were simulated in systems containing litter needles inoculated with individual saprotrophic fungal strains and their mixtures. Fungal strains of Setulipes androsaceus (L.) Antonin, Chalara longipes (Preus) Cooke, Ceuthospora pinastri (Fr.) Hohn., Mollisia minutella (Sacc.) Rehm, Scleroconidioma sphagnicola Tsuneda, Currah & Thormann and an unknown strain NK11 were used as representatives of autochthonous mycoflora. Systems were incubated for 5.5 months in laboratory conditions. Fungal colonization in systems and competition among strains were assessed using the reisolation of fungi from individual needles. After incubation, needles were extracted with NaOH and extracts were analysed using 31 P nuclear magnetic resonance spectroscopy (NMR). Needle decomposition was determined based on the decrease in C:N ratio. Systems inoculated with the basidiomycete S. androsaceus revealed substantial decrease in C:N ratio (from 25.8 to 11.3) while the effect of ascomycetes on the C:N ratio was negligible. We suppose that tested strains of saprotrophic ascomycetes did not participate substantially in litter decomposition, but were directly involved in phosphorus transformation and together with S. androsaceus could transform orthophosphate monoesters and diesters from spruce litter needles into diphosphates, polyphosphates and phosphonates. These transformations seem to be typical for saprotrophic fungi involved in litter needle decomposition, although the proportion of individual phosphorus forms differed among studied fungal strains. Phosphonate presence in needles after fungal inoculation is of special interest because no previous investigation recorded phosphonate synthesis and accumulation by fungi. Our results confirmed that the 31 P NMR spectroscopy is an excellent instrumental method for studying transformations of soil organic phosphorus during plant litter decomposition. We suggest that polyphosphate production by S. androsaceus may contribute to the phosphorus cycle in forest ecosystems because this fungus is a frequent litter colonizer that substantially participates in decomposition.

Indirect spin-spin NMR 1 H- 1 H coupling constants of newly synthesized furanose monosaccharide derivatives were interpreted on the basis of ab initio modeling. Epoxy, epithio, and epimino groups were inserted into the sugars and significantly limited their conformational flexibility, which was confirmed by a systematic conformer analysis. Because of the restriction, the performance of the computations and the dependence of the coupling constants on the geometry could be estimated more easily. Conventional Karplus equations are not optimized for this class of compounds and cannot be used for reliable interpretation of the NMR spectra. Fully analytical B3LYP/IGLOII computations of the coupling constants were performed including all the four important magnetic terms (SD, DSO, PSO, FC) in the Hamiltonian. Good agreement of the calculated and the experimental coupling constants was achieved, and computed structural parameters are consistent with available X-ray data. The influence of the different functional groups on the spin -spin coupling constants was discussed.

Using 1 H, 13 C, 19 F, 31 P, and 14 N NMR spectroscopy, it has been demonstrated that 2-(dimethylamino)ethyl-dimethylphosphoramidofluoridate undergoes spontaneous isomerization to dimethylaziridinium-dimethylamidofluorophosphate.

The assembly of Mason-Pfizer monkey virus Gag polyproteins into immature capsids and their cleavage by the encoded protease are temporally and spatially separated processes, making the virus a particularly useful model for investigation of protease activation. Here we present a high resolution NMR structure of a fully folded monomer of a 12 kDa M-PMV protease (wt 12 PR) and of a Cys7Ala/Asp26Asn/Cys106Ala mutant (12 PRD26N/C7A/C106A). The overall structures of both wt 12 PR and 12 PRD26N/C7A/C106A follow the conservative structural motif of other retroviral proteases. The most prominent difference from the canonical fold of retroviral proteases is the absence of the interfacial β-sheet, which leads to the loss of the principal force stabilizing the dimer of M-PMV PR. The monomer–dimer equilibrium can be shifted in favor of the dimer by adding a substrate or an inhibitor, partially compensating for the missing role of the β-sheet. We also show that cysteines C7 and C106 play a crucial role in stabilizing the dimer and consequently increasing the proteolytic activity of M-PMV PR. This is consistent with the role of reversible oxidative modification of the cysteine residues in the regulation of the maturation of assembled M-PMV capsids in the cytoplasm.

Half-lives and fluorine atom shifts of stabilized 1-fluoro-1-lithioethenes bearing hydrogen, fluorine, phenyl, and/or dimethylphenylsilyl groups in the β-positions have been determined by a low-temperature 19F NMR spectroscopy. Some 1-fluoro-1-lithioethenes displayed an exceptionally low value of the trans-3JFF coupling constant. Stereoselectivity of carbenoid formation, as well as an effect of configuration on the stability is discussed.

Background Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core – a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a β-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the β-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. Results Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the β-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal β-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. Conclusion Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the β-hairpin in mature M-PMV CA. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0094-8) contains supplementary material, which is available to authorized users.

25,26,27,28-Tetrapropoxy-2,8,14,20-tetrathiacalix[4]arene undergoes a thermal equilibration at elevated temperature yielding a mixture of conformers. The rate and equilibrium constants of this process were established using NMR spectroscopy. For the first time the equilibrating process was also used on a preparative scale for the isolation and characterisation of all four basic thiacalix[4]arene conformations.

The conformational behaviour of 25,27-dimethoxythiacalix[4]arene was studied using NMR techniques and X-ray analysis. The title compound prefers a cone conformation in solution, while in the solid state it adopts a unique 1,2-alternate conformation thus creating a novel type of molecular channel held together by π–π interactions.

Chiral pyran derivatives were prepared by hetero-Diels– Alder reaction between ethyl vinyl ether and 1-oxa-1,3-butadienes bearing a chiral substituent in position 4 to obtain more information about the stereoselectivity of this reaction. The structure of reaction products was studied by one-dimensional NMR methods based on a double pulsed field gradient spin echo. Two novel NMR pulse sequences based on a combination of COSY and NOESY/ROESY are described, relieving the problem of heavy overlaps of target resonances in NOE experiments. The NMR studies in combination with further synthetic transformations allowed us to assign the configurations of the isolated cycloadducts. The results indicate that the configuration of the chiral substituent in position 4 is not the principal factor which controls the approach of the ethyl vinyl ether to the planar 1-oxa-1,3-butadiene system. Copyright © 2000 John Wiley & Sons, Ltd.

Some 1-(2-substituted phenyl)- and 1-(1-naphthyl)-2,4,6-triphenylpyridinium perchlorates were treated with potassium hexacyano ferrate(III)-potassium hydroxide reagent to give the title pyrroles. Restricted rotation around the C-N bond in the products is demonstrated by NMR experiments with homochiral shift reagents, by semiempirical PM3 calculations as well as using their atropodiastereoselective and enantioselective transformations. Racemisation barriers for the (R)-2-(N-methyl-N-phenylcarbamoyl) derivative were estimated from NMR and polarimetric measurements.

Treatment of 2,5:3,6-dianhydro-6-thio-4- O -trifluoromethanesulfonyl- l -talose ethylene acetal ( 5 ) with lithium benzoate in boiling DMF unexpectedly gave the 9-crown-3 ether derivatives 7 and 8 instead of the substitution product 6 . The mechanism of the process presumably involved neighbouring group participation of the dioxolane acetal function. 1 H NMR and molecular mechanics calculations (MM3) provided the assignment of stereoisomer configuration since the results of semi-empirical PM3 calculations on postulated oxonium-ion intermediates reasonably explained the high stereoselectivity of the process.

Conformational analysis of a novel tetraethylether of thiacalix[4]arene by means of NMR spectroscopy is presented. Equilibrium between three dominant conformers paco, 1,3-alt and cone exists in CDCl 3 solution at room temperature. Observed chemical exchange between conformers indicates higher internal flexibility of the title compound in comparison with similar methylene bridged analogues of tetraethylethers of calix[4]arene and p- tert -butylcalix[4]arene. The cone conformer experiences additional internal motions.

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.

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)

We present the preparation of the xenotropic murine leukaemia virus-related virus matrix protein (XMRV-MA) and its structure determined by NMR spectroscopy. The DNA fragment encoding XMRV-MA was obtained from prostate tumour cell cDNA (Rv1 cell line) by PCR and inserted into a pET-22b plasmid. Non-myristoylated, uniformly 13C- and 15N-labeled XMRV-MA, fused with histidine tag, was produced in E. coli BL21 (DE3) cells. The protein was purified by immobilized metal affinity chromatography (NiNTA-agarose) and size-exclusion chromatography (Sephadex 75), and then concentrated to 5 mg/ml. All NMR data were collected at 298 K on a 600 MHz Bruker Avance III spectrometer equipped with a cryogenic triple-resonance probe and analyzed with CcpNmr Analysis. Back-bone and side-chain resonances were assigned using standard NMR experiments and structural constraints were obtained from 13C- and 15N-edited NOESY experiments. Structures were calculated with ARIA. Although the protein sequence of the XMRV-MA is very similar to that of the murine leukaemia virus matrix protein (MLV-MA), it varies in several amino acid residues. We compared the structures of the XMRV-MA and MLV-MA and found that those changes are localized in a few domains, mostly on the surface of the protein.

The oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural preorganization play an important role in the control of the oligomerization process.

Irradiation of octafluoro-1,3,5-hexatriene ( I ) in the gas phase with a high-pressure mercury lamp gave a mixture of octafluoro-2-vinylcyclobutene ( III ) and octafluorobicyclo[2.2.0]hex- 2-ene ( V ). The analogous photolysis of hexatriene I in the liquid phase led to an equilibrium mixture of the starting compound I ( cis -isomer) and the trans -isomer II , respectively. Photolysis of 2,3,4,5-tetrafluoro-1,3,5-hexatriene ( VI ) gave a mixture of cis - and trans - isomers, both in the vapour and liquid phases. Thermal reaction of hexatriene I gave octafluoro-1,3-cyclohexadiene ( IV ) as the sole product.

The reaction of 2-cinnamoylfluorene II with quaternary pyridinium salts IIIa, IIIb in the presence of ammonium acetate, gave 2-fluorenyl-2,6-diarylpyridines IV. The complete assignment of 1H and 13C resonances by 2D NMR methods is given.

We studied the oligomeric properties of betaretroviral nonmyristoylated matrix protein (MA) and its R55F mutant from the Mason–Pfizer monkey virus in solution by means of chemical crosslinking and NMR spectroscopy. By analyzing crosslinked products and using concentration-dependent NMR chemical shift mapping, we have proven that the wild-type (WT) MA forms oligomers in solution. Conversely, no oligomerization was observed for the R55F mutant. Structural comparison of MAs explained their different behaviors in solution, concluding that the key residues involved in intermonomeric interaction are exposed in the WT MA but buried in the mutant, preventing the oligomerization of R55F. The final model of oligomerization of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason–Pfizer monkey virus WT MA exists in a monomer–dimer–trimer equilibrium in solution, with the corresponding dissociation constants of 2.3 and 0.24 mM, respectively. Structures of the oligomers calculated with HADDOCK software are closely related to the structures of other retroviral MA trimers.

The stereoselectivity of the reaction of furan (1) with maleic anhydride (2) and maleimide (3) was studied experimentally and theoretically. Although the two reactions are highly similar with regard to their preference for endo and exo steroisomers, notable differences were experimentally observed and explained on the basis of calculated reaction-free energies and transition-state barriers. The experimental values of rate constants (k(1+2endo) = (1.75 +/- 0.48) x 10(-5); mol(-1) l s(-1); k(1+2exo) = (3.10 +/- 0.55) x 10(-5); mol(-1) l s(-1); k(1+3endo) = (1.93 +/- 0.082) x 10(-5); mol(-1) l s(-1), k(1+3exo) = (1.38 +/- 0.055) x 10(-5); mol(-1) l s(-1) all at 300 K) and the observed reaction course clearly confirm that neither of these reactions are prototypical examples of Diels-Alder [4 + 2] cycloadditions, whose dominant preference is for endo isomers. However, only by comparing their energetics-calculated at the CCSD(T) level of theory-with the analogous reactions involving cyclopentadiene (8) as a diene can these observations be understood. The low thermodynamic stability of furan [4 + 2] adducts opens retro-Diels-Alder reaction channels and overrules the very small kinetic preference (calculated and measured here) of initial formation for endo stereoisomers. On a macroscopic scale "an irregular"-thermodynamically more stable-exo stereoisomer was consequently observed as a dominant species.

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.

Formation of trimers of the light-harvesting complex II (LHCII) of plant thylakoid membranes is dependent on the presence of the lipid phosphatidylglycerol (PG) [1,2] and on the presence of certain conserved amino acid residues in the N-terminal [3] and C-terminal [4] region. Hobe et al. [3] presented hypothesis that region 16 – 21 is the PG binding site. LHCII is one of dominant phosphoproteins of thylakoids [5]. Phosphorylation site is located in the neighbourhood of the trimerisation site and is sequentially highly variable [6]. Upon phosphorylation of LHCII are exposed to solution aromatic amino acid sidechains [7] substantial part of which is located in the putative lipid binding site. The known structure of LHCII [8,9] does not give any details about the periphery of the extra-membrane domain where the N-terminus of LHCII extends. Jansson et al. [10] found that Lhcb2 subunits are more abundant in granal fractions than in stromal fractions. PG is more abundant than in photosystem II membranes than is its average concentration in thylakoids [11], while the distribution of other lipids is more or less uniform. Lipids together with LHCII cover most of the membrane surface and thus determine its properties. Subsequently, the surface properties determine both the adhesion of membrane lamellae and the separation of membrane fractions.

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.

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.

(3,5-Diphenyl-1-o-tolyl-1H-pyrrol-2-yl)phenylmethanone 3a and (1-naphthalen-1-yl-3,5-diphenyl-1H-pyrrol-2-yl)phenylmethanone 4a are atropisomeric and their LAH reductions afford mixtures of atropdiastereoisomeric (3,5-diphenyl-1-o-tolyl-1H-pyrrol-2-yl)-phenylmethanoles 3b,c and (1-naphthalen-1-yl-3,5-diphenyl-1H-pyrrol-2-yl)phenylmethanoles 4b,c, respectively.