A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phosph... more A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.
The new anionic carbon sulfides C 6 S 10 2and C 12 S 16 2are described and crystallographically c... more The new anionic carbon sulfides C 6 S 10 2and C 12 S 16 2are described and crystallographically characterized. The C 12 S 16 2anion consists of two C 6 S 8 units connected by an exceptionally long (2.157(12) Å) S-S bond. In solution, C 12 S 16 2exists in equilibrium with the radical C 6 S 8-•. The equilibrium constant for radical formation (293 K, THF) is 1.2 × 10-4 M, as determined by optical spectroscopy at varying concentrations. Radical formation occurs through scission of the S-S bond. On the basis of variable temperature EPR spectra, the thermodynamic parameters of this process are ∆H) +51.5 (0.5 kJ mol-1 and ∆S) +110 (3 J mol-1 K-1. C 6 S 10 2is an oxidation product of C 3 S 5 2and consists of two C 3 S 5 units connected by an S-S bond. The S-S bond length (2.135(4) Å) is long, and the CS-SC torsion angle is unusually acute (52.1°), which is attributed to an attractive interaction between C 3 S 2 rings. The oxidation of (Me 4 N) 2 C 3 S 5 occurs at-0.90 V vs Fc + /Fc in MeCN, being further oxidized at-0.22 V. The similarity of the cyclic voltammogram of (Me 4 N) 2 C 6 S 10 to that of (Me 4 N) 2 C 3 S 5 indicates that C 6 S 10 2is the initial oxidation product of C 3 S 5 2- .
The new anionic carbon sulfides C 6 S 10 2and C 12 S 16 2are described and crystallographically c... more The new anionic carbon sulfides C 6 S 10 2and C 12 S 16 2are described and crystallographically characterized. The C 12 S 16 2anion consists of two C 6 S 8 units connected by an exceptionally long (2.157(12) Å) S-S bond. In solution, C 12 S 16 2exists in equilibrium with the radical C 6 S 8-•. The equilibrium constant for radical formation (293 K, THF) is 1.2 × 10-4 M, as determined by optical spectroscopy at varying concentrations. Radical formation occurs through scission of the S-S bond. On the basis of variable temperature EPR spectra, the thermodynamic parameters of this process are ∆H) +51.5 (0.5 kJ mol-1 and ∆S) +110 (3 J mol-1 K-1. C 6 S 10 2is an oxidation product of C 3 S 5 2and consists of two C 3 S 5 units connected by an S-S bond. The S-S bond length (2.135(4) Å) is long, and the CS-SC torsion angle is unusually acute (52.1°), which is attributed to an attractive interaction between C 3 S 2 rings. The oxidation of (Me 4 N) 2 C 3 S 5 occurs at-0.90 V vs Fc + /Fc in MeCN, being further oxidized at-0.22 V. The similarity of the cyclic voltammogram of (Me 4 N) 2 C 6 S 10 to that of (Me 4 N) 2 C 3 S 5 indicates that C 6 S 10 2is the initial oxidation product of C 3 S 5 2- .
Biochimica Et Biophysica Acta - General Subjects, Jul 1, 1994
A new technique to study cellular respiration under steady-state conditions is described. For det... more A new technique to study cellular respiration under steady-state conditions is described. For detection of oxygen concentration the technique utilizes lithium phthalocyanine crystals whose, electron paramagnetic resonance (EPR) spectra are highly sensitive to oxygen. A gas permeable poly(tetrafluoroethylene) (TFE) capillary (i.d. = 0.81 mm; o.d. = 0.86 mm) is used to control the oxygen flux to the sample of cultured neural cells (N1E-115) of 50 ~1 volume. The measured oxygen permeability of the TFE capillary exhibits Arrhenius law behavior (Ep = 21.2 kJ/mole) in the temperature range 15-43°C. The observed increase in the respiratory rate of the N1E-115 cells with temperature can be described with an apparent activation energy of E~ = 110 _+ 20 KJ/mol. Cellular depolarization with 35 mM KCI increases the respiratory rate of the cells but gives the same activation energy. The results confirm a hypothesis that respiratory depolarizers affect only the total enzyme concentration and do not change the apparent E a of the substrate-enzyme decomposition reaction. The theoretical treatment of oxygen diffusion in the system provides guidelines for designing steady-state cellular respiration experiments.
High-field EPR provides significant advantages for studying structure and dynamics of molecular s... more High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with nonresonant absorption by water and other polar molecules. The strong absorbance of mmW's by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Qfactor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E=0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was ≈520. The Q-factor decreased slightly to ≈450 when loaded with a water-containing nanoporous disc of 50 μm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE 012-type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators.
This communication reports the first example of a high resolution solid-state 15 N 2D PISEMA NMR ... more This communication reports the first example of a high resolution solid-state 15 N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15 N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15 N and 31 P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.
A novel method for studying membrane proteins in a native lipid bilayer environment by solid-stat... more A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60-mm-long nanopores were employed to form macroscopically aligned peptide-containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a period of up to a few hours and temperatures as high as 70°C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid-state 19.6 T 17 O NMR study of reversible binding effects of mono-and divalent ions on the chemical shift properties of the Leu 10 carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the 17 O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMRbased studies, including structure-function, drug screening, proton exchange, pH, and other titration experiments, will benefit significantly from the method described here.
Bulletin of the American Physical Society, Mar 16, 2017
in real time by means of an in situ Quartz Crystal Microbalance (QCM) technique, with a focus on ... more in real time by means of an in situ Quartz Crystal Microbalance (QCM) technique, with a focus on the impact of the suspension on the surface roughness and texture of the QCM electrode and how the results compared to macroscopic reductions in friction and increased energy efficiency for the same materials' combinations. The frequency and dissipative properties (mechanical resistance) of QCM's with both gold and nickel surface electrodes were first studied for immersed samples upon addition of the nanoparticles. Nanodiamonds resulted in an increased mechanical resistance while the addition of Al 2 O 3 and SiO 2 nanoparticles resulted in a decreased resistance, indicating a reduced resistance of the fluid to the motion of the QCM. Atomic Force Microscope (AFM) measurements were then performed on the QCM electrodes after exposure to the suspensions, to explore potential polishing and/or roughening effects. The results are closely linked to the macroscopic friction and wear attributes.
The Metabolome of Food Knowledge Database: Development of a Nutrition Database to Support Precision Nutrition
Current developments in nutrition, Jun 1, 2022
Objectives To develop a precision nutrition knowledge database, with the aim to provide individua... more Objectives To develop a precision nutrition knowledge database, with the aim to provide individualized and actionable dietary recommendations to help prevent disease. However presently, dietary phytochemicals are poorly represented in current metabolomic databases. To address this gap, we are building a cloud-based knowledge database (KDB) named “The Metabolome of Food” (MetaboFood®) which focuses on phytochemical compositional, metabolite and pathway data. Methods MetaboFood® features P-MetDB®, a database in static tabular form, of nutritionally relevant phytochemicals and their metabolites derived from systematic literature reviews of 17 commonly consumed phytochemical-rich foods, matched to InChI key, physical and chemical properties (mass, formula) and database identifiers (i.e., PubChem ID, KEGG ID, SMILES etc.). To build MetaboFood®, information about metabolic pathways and diseases associated with these foods have been extracted from various pathway databases using APIs that these databases provide. Information can be searched in MetaboFood® and results are explored in a highly visual and interactive way, in the form of self-organizing maps, node-link diagrams, Sankey diagrams and other visual analytics techniques. Results MetaboFood® captures data on foods, their phytochemical compositions, human and microbial metabolites, and pathway and diseases linkages. Information in MetaboFood® facilitates both hypothesis generation and hypothesis testing relative to food and pathway analysis. Initial use of this database identifies significant interactions between polyphenol rich foods and numerous metabolic networks. Conclusions MetaboFood® builds on traditional food composition databases by integrating biochemical and disease pathway data with diet metabolites. A key to moving forward is building data richness, enabling greater connections between diet and health. Funding Sources Research reported in this abstract was supported by a NIEHS Human Health Exposure Analysis Resource (HHEAR) program grant under award number 1U2CES030857-01 and a NIH Nutrition for Precision Health (NPH) Metabolomics and Clinical Assay Center (MCAC) grant under the award number 1U24CA268153-01. CDK was also supported by the USDA National Institute of Food and Agriculture Hatch award (Kay-Colin; 1,011,757).
Electron irradiation-induced paramagnetic and fluorescent defects in type Ib high pressure–high temperature microcrystalline diamonds and their evolution upon annealing
Journal of Applied Physics, Aug 17, 2022
Defects introduced to synthetic type Ib diamond micrometer-size particles by electron-beam irradi... more Defects introduced to synthetic type Ib diamond micrometer-size particles by electron-beam irradiation were studied by electron paramagnetic resonance and photoluminescence (PL) spectroscopy as a function of e-beam fluence and post-irradiation thermal annealing. Increasing electron-beam fluence causes a substantial reduction of the substitutional nitrogen (P1) content, accompanied by progressively higher concentrations of paramagnetic negatively charged vacancies (V−) and triplet interstitials (R1/R2). Annealing results in a drastic decrease in the V− and R1/R2 content and an increase in the negatively charged nitrogen-vacancies (NV− or W15). Analysis of PL spectra allows for identification of color centers in the irradiated diamond samples and following their evolution after annealing. These data facilitate understanding of different factors contributing to the formation of color centers in diamond and promote efforts toward controlled engineering of optical centers in fluorescent diamond particles.
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