Spectroscopic Study of the Basicity of 4,6-Dihydroxypyrimidine Derivatives.

The protonation of a number of 4,6-dihydroxypyrimidine derivatives is studied, and the features of the electronic spectra of free bases and protonated forms are considered. It is shown that the alkyl substituents in position 2 increase the basicity of the compound, and the nitro group in position 5 leads to its decrease. In an acid medium (0.1-99.5% H2SO4), 4,6-dihydroxypyrimidine, 6-hydroxy-2-methylpyrimidine-4(3H)-one, and 6-hydroxy-2-ethylpyrimidine-4(3H)-one have two protonation stages, barbituric acid is protonated in three stages, and 6-hydroxy-2-methyl-5-nitropyrimidine-4(3H)-one and 6-hydroxy-2-ethyl-5-nitropyrimidine-4(3H)-one form a monocation.

CharmeRT: Boosting Peptide Identifications by Chimeric Spectra Identification and Retention Time Prediction.

Coeluting peptides are still a major challenge for the identification and validation of MS/MS spectra, but carry great potential. To tackle these problems, we have developed the here presented CharmeRT workflow, combining a chimeric spectra identification strategy implemented as part of the MS Amanda algorithm with the validation system Elutator, which incorporates a highly accurate retention time prediction algorithm. For high-resolution data sets this workflow identifies 38-64% chimeric spectra, which results in up to 63% more unique peptides compared to a conventional single search strategy.

Probing the interaction of the potassium channel modulating KCNE1 in lipid bilayers via solid-state NMR spectroscopy.

KCNE1 is known to modulate the voltage-gated potassium channel α subunit KCNQ1 to generate slowly activating potassium currents. This potassium channel is essential for the cardiac action potential that mediates a heartbeat as well as the potassium ion homeostasis in the inner ear. Therefore, it is important to know the structure and dynamics of KCNE1 to better understand its modulatory role. Previously, the Sanders group solved the three-dimensional structure of KCNE1 in LMPG micelles, which yielded a better understanding of this KCNQ1/KCNE1 channel activity. However, research in the Lorigan group showed different structural properties of KCNE1 when incorporated into POPC/POPG lipid bilayers as opposed to LMPG micelles. It is hence necessary to study the structure of KCNE1 in a more native-like environment such as multi-lamellar vesicles. In this study, the dynamics of lipid bilayers upon incorporation of the membrane protein KCNE1 were investigated using 31 P solid-state nuclear magnetic resonance (NMR) spectroscopy. Specifically, the protein/lipid interaction was studied at varying molar ratios of protein to lipid content. The static 31 P NMR and T1 relaxation time were investigated. The 31 P NMR powder spectra indicated significant perturbations of KCNE1 on the phospholipid headgroups of multi-lamellar vesicles as shown from the changes in the 31 P spectral line shape and the chemical shift anisotropy line width. 31 P T1 relaxation times were shown to be reversely proportional to the molar ratios of KCNE1 incorporated. The 31 P NMR data clearly indicate that KCNE1 interacts with the membrane. Copyright © 2017 John Wiley & Sons, Ltd.

International Occupational Therapy Research Priorities.

Occupational therapy is a global profession represented by the World Federation of Occupational Therapists (WFOT). International research priorities are needed for strategic guidance on global occupational therapy practice. The objective of this study was to develop international research priorities to reflect global occupational therapy practice. A Delphi study using three rounds of electronic surveys, distributed to WFOT member organizations and WFOT accredited universities, was conducted. Data were analyzed after each round, and priorities were presented for rating and ranking in order of importance. Forty-six (53%) out of 87 WFOT member countries participated in the Delphi process. Eight research priorities were confirmed by the final electronic survey round. Differences were observed in rankings given by member organizations and university respondents. Despite attrition at Round 3, the final research priorities will help to focus research efforts in occupational therapy globally. Follow-up research is needed to determine how the research priorities are being adopted internationally.

Solid-state NMR (31)P paramagnetic relaxation enhancement membrane protein immersion depth measurements.

Paramagnetic relaxation enhancement (PRE) is a widely used approach for measuring long-range distance constraints in biomolecular solution NMR spectroscopy. In this paper, we show that (31)P PRE solid-state NMR spectroscopy can be utilized to determine the immersion depth of spin-labeled membrane peptides and proteins. Changes in the (31)P NMR PRE times coupled with modeling studies can be used to describe the spin-label position/amino acid within the lipid bilayer and the corresponding helical tilt. This method provides valuable insight on protein-lipid interactions and membrane protein structural topology. Solid-state (31)P NMR data on the 23 amino acid α-helical nicotinic acetylcholine receptor nAChR M2δ transmembrane domain model peptide followed predicted behavior of (31)P PRE rates of the phospholipid headgroup as the spin-label moves from the membrane surface toward the center of the membrane. Residue 11 showed the smallest changes in (31)P PRE (center of the membrane), while residue 22 shows the largest (31)P PRE change (near the membrane surface), when compared to the diamagnetic control M2δ sample. This PRE SS-NMR technique can be used as a molecular ruler to measure membrane immersion depth.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

Solid-state NMR investigations of peptide-lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106.

The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro-Gly hinge, an amphipathic α-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, (31)P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide-lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid.

Structure-property relationships of a biological mesocrystal in the adult sea urchin spine.

Structuring over many length scales is a design strategy widely used in Nature to create materials with unique functional properties. We here present a comprehensive analysis of an adult sea urchin spine, and in revealing a complex, hierarchical structure, show how Nature fabricates a material which diffracts as a single crystal of calcite and yet fractures as a glassy material. Each spine comprises a highly oriented array of Mg-calcite nanocrystals in which amorphous regions and macromolecules are embedded. It is postulated that this mesocrystalline structure forms via the crystallization of a dense array of amorphous calcium carbonate (ACC) precursor particles. A residual surface layer of ACC and/or macromolecules remains around the nanoparticle units which creates the mesocrystal structure and contributes to the conchoidal fracture behavior. Nature's demonstration of how crystallization of an amorphous precursor phase can create a crystalline material with remarkable properties therefore provides inspiration for a novel approach to the design and synthesis of synthetic composite materials.

Chemical synthesis of polyprenyl sialyl phosphate, a probable biosynthetic intermediate of bacterial polysialic acid.

Using reaction of moraprenyl phosphate with the known N-acetylsialyl chloride and the novel N,N-diacetylsialyl (Neu5Ac(2)) chloride α- and ß-anomers of polyprenyl sialyl phosphate were synthesized for the first time. The α-selectivity dramatically increased when Neu5Ac(2) chloride was used as the glycosyl donor.

Membrane proteins structure and dynamics by nuclear magnetic resonance.

Membrane proteins represent a challenging class of biological systems to study. They are extremely difficult to crystallize and in most cases they retain their structure and functions only in membrane environments. Therefore, commonly used diffraction methods fail to give detailed molecular structure and other approaches have to be utilized to obtain biologically relevant information. Nuclear magnetic resonance (NMR) spectroscopy, however, can provide powerful structural and dynamical constraints on these complicated systems. Solution- and solid-state NMR are powerful methods for investigating membrane proteins studies. In this work, we briefly review both solution and solid-state NMR techniques for membrane protein studies and illustrate the applications of these methods to elucidate proteins structure, conformation, topology, dynamics, and function. Recent advances in electronics, biological sample preparation, and spectral processing provided opportunities for complex biological systems, such as membrane proteins inside lipid vesicles, to be studied faster and with outstanding quality. New analysis methods therefore have emerged, that benefit from the combination of sample preparation and corresponding specific high-end NMR techniques, which give access to more structural and dynamic information.

Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers.

A new approach for determining the membrane immersion depth of a spin-labeled probe has been developed using paramagnetic relaxation enhancement (PRE) in solid-state NMR spectroscopy. A DOXYL spin label was placed at different sites of 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) phospholipid bilayers as paramagnetic moieties and the resulting enhancements of the longitudinal relaxation (T1) times of ³¹P nuclei on the surface of the bilayers were measured by a standard inversion recovery pulse sequence. The ³¹P NMR spin-lattice relaxation times decrease steadily as the DOXYL spin label moves closer to the surface as well as the concentration of the spin-labeled lipids increase. The enhanced relaxation vs. the position and concentration of spin-labels indicate that PRE induced by the DOXYL spin label are significant to determine longer distances over the whole range of the membrane depths. When these data were combined with estimated correlation times τ(c), the r⁻6-weighted, time-averaged distances between the spin-labels and the ³¹P nuclei on the membrane surface were estimated. The application of using this solid-state NMR PRE approach coupled with site-directed spin labeling (SDSL) may be a powerful method for measuring membrane protein immersion depth.

Prenyl sulfates as alkylating reagents for mercapto amino acids.

A new methodology for prenylation of thiol compounds has been developed. The approach is based on the use of prenyl sulfates as new reagents for S-prenylation of benzenethiol and cysteamine in aqueous systems. The C(10)-prenols geraniol and nerol that differ in the configuration (E or Z, correspondingly) of the alpha-isoprene unit were efficiently O-sulfated in the presence of a pyridine-SO(3') complex. The obtained geranyl and neryl sulfates were tested as alkylating agents. These compounds were chosen to reveal the influence of the alpha-isoprene unit configuration on their alkylation (prenylation) ability. S-Geranyl cysteine was prepared to demonstrate the applicability of this method for prenylation of peptides containing mercapto amino acids.

Synthesis of a quaternary polyprenyl ammonium salt.

Reaction of primary C(55)-allylic alcohol moraprenol (WT(3)C(7-9)-OH, a polyprenol from mulberry leaves) with triethylamine in the presence of phosphorus oxychloride leads to a quaternary ammonium chloride with a good yield (72%) and high cis-stereoselectivity of the terminal isoprene unit. Cationic polyprenyl derivatives may be useful for transfection and immunological studies.

A novel software tool for copolymer characterization by coupling of liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The results of copolymer characterization by coupling of chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques and subsequent calculation of copolymer composition using a novel software tool 'MassChrom2D' are presented. For high-resolution mass analysis copolymer samples were fractionated by means of liquid adsorption chromatography (LAC). These fractions were investigated off-line by MALDI-TOF MS. Various mono-n-butyl ethers of polyethylene oxide-polypropylene oxide copolymers (PEO-co-PPO) were investigated. As well as the copolymer composition presented in two-dimensional plots, the applied approach can give additional hints on specific structure-dependent separation conditions in chromatography.

A novel three-component reaction designed by the combinatorial method: heteroarenes, isothiocyanates and isocyanides.

The novel three-component reaction of isoquinoline with isothiocyanates and isocyanides leads to a variety of new imidazoisoquinolines. The zwitterionic ground state of these new ring systems is established by means of NMR and X-ray analysis. Use of phthalazine instead of isoquinoline gives access to imidazole annulated phthalazines.