Natural Abundance Carbon-13 Research Articles

New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles

Galactolipids are the main structural component of plant chloroplastic (thylakoid) membranes and of blue-green algae cell membranes. The predominant lipids in this class are monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG). We here present a method for the preparation of bicelles that contain these galactolipids together with a characterization of the bicelles, and the lipids within the bicelles. NMR diffusion data show that up to 30% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a q = 0.5 DMPC/DHPC lipid matrix can be replaced with either monogalactosyl-diacylglycerol or digalactosyl-diacylglycerol and that these lipids incorporate into the bicelles. No evidence for phase separation is observed. Bicelles made with monogalactosyl-diacylglycerol are significantly larger than bicelles containing only DMPC, already with only 10% of the DMPC replaced with the galactolipid. The effect of digalactosyl-diacylglycerol on bicelle size is much smaller. These observations are likely to be correlated with the different bilayer-forming properties of the lipids. Monogalactosyl-diacylglycerol is a non-bilayer-forming lipid, while digalactosyl-diacylglycerol is a bilayer-forming lipid. Both galactolipids display extensive local motion within the bilayer, as evidenced by natural abundance carbon-13 relaxation of the lipid molecules. The sugar headgroup regions are motionally restricted and cannot be described by a model that does not take into account anisotropic reorientation of the sugar units. No significant effect of the galactolipids on DMPC dynamics was observed. Our results indicate that these bicelles may become useful as model membrane mimetic media for studies of galactolipid-protein interactions.

RαStructures of partially oriented pentafluorobenzenes C6F5X (X = H, Cl, I) as determined from19F NMR spectra with13C satellites

The (19)F spectra with natural-abundance carbon-13 satellites of C(6)F(5)X (X = H, Cl or I) in ZLI 1695 liquid crystal were analysed. Excluding fluorine-fluorine dipolar coupling constants from the fitting, the vibrationally corrected structures of these molecules were derived and compared with those calculated at DFT/B3LYP level of theory with the aug-cc-pVTZ basis set. The results show that pentafluorobenzene did not exhibit noticeable distortion of the aromatic ring, while chloropentafluorobenzene and iodopentafluorobenzene molecules showed some deformations in their determined structures. Relative anisotropies of F-F couplings have been deduced with accuracy for C(6)F(5)H molecule. Due to uncertainties in structure determination of C(6)F(5)Cl and C(6)F(5)I molecules, it was not possible to obtain precise values for all the corresponding anisotropies. In addition, it was found that the orientation of these molecules in the solvent used can be qualitatively explained in terms of dispersion forces.

Long-lived states to sustain hyperpolarized magnetization.

Major breakthroughs have recently been reported that can help overcome two inherent drawbacks of NMR: the lack of sensitivity and the limited memory of longitudinal magnetization. Dynamic nuclear polarization (DNP) couples nuclear spins to the large reservoir of electrons, thus making it possible to detect dilute endogenous substances in magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). We have designed a method to preserve enhanced ("hyperpolarized") magnetization by conversion into long-lived states (LLS). It is shown that these enhanced long-lived states can be generated for proton spins, which afford sensitive detection. Even in complex molecules such as peptides, long-lived proton states can be sustained effectively over time intervals on the order of tens of seconds, thus allowing hyperpolarized substrates to reach target areas and affording access to slow metabolic pathways. The natural abundance carbon-13 polarization has been enhanced ex situ by almost four orders of magnitude in the dipeptide Ala-Gly. The sample was transferred by the dissolution process to a high-resolution magnet where the carbon-13 polarization was converted into a long-lived state associated with a pair of protons. In Ala-Gly, the lifetime T(LLS) associated with the two nonequivalent H(alpha) glycine protons, sustained by suitable radio-frequency irradiation, was found to be seven times longer than their spin-lattice relaxation time constant (T(LLS)/T(1) = 7). At desired intervals, small fractions of the populations of long-lived states were converted into observable magnetization. This opens the way to observing slow chemical reactions and slow transport phenomena such as diffusion by enhanced magnetic resonance.

Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides

The morphology of q = 0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.

Salinity-induced Patterns of Natural Abundance Carbon-13 and Nitrogen-15 in Plant and Soil

Salinity-induced Patterns of Natural Abundance Carbon-13 and Nitrogen-15 in Plant and Soil

NMR Spectra of Nitrobenzanthrones Obtained by Using Double-Pulsed Field-Gradient Spin Echo

We have synthesized several nitrobenzanthrones (NBAs) and investigated their structures in solution using NMR spectroscopy. In our study, however, low solubility of NBAs in organic solvents always became a serious problem and led to NMR spectra with low signal-to-noise ratios. To solve this problem, we adopted a new method of conventional coherence transfer pathway selection using pulsed field-gradients. The double-pulsed field-gradient spin echo (DPFGSE) sequence was used for selecting natural-abundance carbon-13 isotopomers by using a bilinear rotation decoupling (BIRD) sequence and a so-called gradient-BIRD pulse for the excitation sculpting technique. We performed NMR measurements on 3,9-dinitro-7 H -benz[ d,e ]anthracene-7-one (compound 1), 3,9,11-trinitro-7 H -benz[ d,e ]anthracene-7-one (compound 2), and 2-nitro-7 H -benz[ d,e ]anthracene-7-one (compound 3) by this method and obtained spectra of high quality. Combining the results from phase-sensitive 2D NOESY, HOHAHA, and DPFGSE-HMQC, DPFGSE-HMQC TOCSY, and phase-sensitive FG-HMBC permitted us to complete NMR assignments. It was confirmed that the present method enables us to improve the sensitivity and to assign the NMR signal readily, even for compounds with low solubility.

Carbon-13 NMR Investigations of the Orientational Order in a Columnar Liquid Crystal

We report a natural abundance carbon-13 nuclear magnetic resonance (NMR) investigation of benzene dissolved in a columnar liquid crystal formed by the carbohydrate mesogen octa-O-decanoyl-β-cellobiose (Cel-II-10). The alignment of the mesophase in the magnetic field is sensitive to the field strength and to the thermal history of the sample. From carbon-13 line shapes, the director distribution functions and the molecular order parameter of the solute were estimated. In contrast to most columnar phases, Cel-II-10 aligns parallel to the magnetic field. The orientational order parameter of benzene is positive, which is expected for a columnar liquid crystal where the symmetry axis of the disk is aligned parallel to the column axis. However, an unexpected increase of the order parameter with increased temperature was observed. This behavior is probably a consequence of an exchange process between different solvation sites in the columnar mesophase and indicates that the population in the environment with higher order parameter increases when the sample is heated.

Phase-Sensitive Two-Dimensional HMQC and HMQC-TOCSY Spectra Obtained Using Double Pulsed-Field-Gradient Spin Echoes

Abstract A new and promising method for recording phase-sensitive HMQC and HMQC-TOCSY spectra of molecules with natural-abundance carbon-13 isotopes is described. It avoids phase modulation of the NMR signals obtained with conventional coherence-transfer pathway selection using pulsed field gradients and results in an improvement in sensitivity. The spectra can be processed according to standard States–Haberkorn–Ruben methods, making the incorporation of gradients transparent to routine users. Strong unwanted proton signals from the carbon-12 isotopomers are attenuated by a double-gradient BIRD pulse at the beginning of the pulse sequences, resulting in high-quality spectra. By incorporating carefully designed purging sequences, high-resolution proton multiplets are obtained.

Measurement and Analysis of the Molecular Ordering Tensors of Two Enantiomers Oriented in a Polypeptide Liquid Crystalline System

High-resolution proton and natural abundance carbon-13 NMR spectra of a rigid chiral molecule, (&)-p(trichloromethy1)-p-propiolactone, dissolved in the liquid-crystalline phase of poly(y-benzyl-L-glutamate) (PBLG), in methylene chloride, were analyzed. From the NMR data and the X-ray structure, the full order matrix was determined and analyzed in detail for each optical isomer of the molecule. It is shown that the spectral discrimination of the enantiomers in this lyotropic medium arises mainly from the difference in their average molecular orientation and the principal axis systems of orientation are compared.

Applications of Natural-Abundance Carbon-13 NMR to Studies of Proteins and Glycoproteins

Applications of Natural-Abundance Carbon-13 NMR to Studies of Proteins and Glycoproteins

Multinuclear NMR spectroscopic studies of some organometallic N-substituted anilines

Proton, carbon-13, nitrogen-15, fluorine-19, and silicon-29 NMR chemical shifts and coupling constants are reported for nine N-substituted anilines of the type XC 6H 4N(H)Y, where X  H and F, and Y  Si(CH 3) 3, Ge(CH 3) 3, Sn(CH 3) 3, and P(BH 3)(CH 3) 2, and the 15N isotopomer of N-trimethylsilylaniline. Resonances in the natural-abundance carbon-13 NMR spectra for both sets of derivatives are assigned on the basis of additivity relationships, proton-coupled spectra, and relative magnitudes of | J( 31P 13C)| and | J( 19F 13C)| coupling constants where appropriate. Carbon-13 and fluorine-19 chemical shifts, and | 1 J( 13C 1H)| coupling constants indicate that the P(BH 3)(CH 3) 2 group is substantially electron-withdrawing.

Assignment of the natural abundance carbon-13 spectrum of proteins using carbon-13-proton detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants. [Erratum to document cited in CA115(19):202475n

Assignment of the natural abundance carbon-13 spectrum of proteins using carbon-13-proton detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants. [Erratum to document cited in CA115(19):202475n

Assignment of the natural abundance carbon-13 spectrum of proteins using carbon-13-proton detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants

Proton-detected heteronuclear multiple-bond 1H-13C correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance 13C spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional 1H assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C alpha H and the carbonyl carbon provides information about the backbone angles psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing 13C NMR spectra of nonenriched proteins. The method yields 13C NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc.

13C-NMR studies of membrane lipid-protein interactions upon protein heat denaturation

Spinach chloroplast membranes were studied by natural abundance carbon-13 nuclear magnetic resonance ( 13C-NMR) spectroscopy in their normal state and after heat denaturation of membrane proteins. The membrane proteins were denatured by raising the temperature of the sample to 67°C for 5 minutes [YashRoy, R.C. (1991) J. Biochem. Biophys. Methods 22, 55–59]. Line-broadening of 13C-NMR resonances arising from the 1st (carbonyl), 7th, 9th and 12th carbon atom of fatty-acyl chains with reference to the carbonyl (C-1) group shows increased immobilization of lipid fatty-acyl chains at these locations, obviously caused by changes in interactions between membrane lipids and proteins upon heat denaturation of membrane proteins.

Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance carbon-13 heteronuclear NMR spectroscopy

Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance carbon-13 heteronuclear NMR spectroscopy

High-pressure nuclear-magnetic-resonance study of carbon-13 relaxation in 2-ethylhexyl benzoate and 2-ethylhexyl cyclohexanecarboxylate

Natural abundance carbon-13 spin-lattice relaxation times and 13G–1H nuclear Overhauser enhancement (NOE) times of 2-ethyl hexylbenzoate (EHB) and 2-ethyl hexylcyclohexanecarboxylate (EHC) have been measured along isotherms of −20, 0, 20, 40, and 80 °C at pressures of 1–5000 bars using high-pressure, high-resolution NMR techniques. The ability to use pressure as an experimental variable has allowed us to study a wide range of molecular motions from extreme narrowing into the slow motional regime. In addition, the high-resolution capability even at high pressure permits the measurement of 13C and NOE for each individual carbon in the molecules studied. Relaxation in both molecules is successfully analyzed in terms of a model assuming a Cole–Davidson distribution of correlation times. The comparison of parameters used in the model demonstrates the increased flexibility of the EHC ring over the EHB ring and also shows how the presence of the flexible ring contributes to the increased over-all mobility of the EHC molecule. The analysis of molecular reorientations in terms of activation volumes also indicates that EHB motion is highly restricted at low temperature.

Simplification of proton-detected, natural abundance carbon-13 correlation spectra of proteins via multiplet editing

Simplification of proton-detected, natural abundance carbon-13 correlation spectra of proteins via multiplet editing

The isolation of an unusual parvalbumin from the white muscle of the silver hake ( Merluccius bilinearis)

Three parvalbumin isotypes have been purified and partially characterized from the skeletal muscle of the silver hake ( Merluccius bilinearis). All three of the isotypes are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins, but the major isotype is distinguishable from the other parvalbumins in that it possesses an unusually high quantity of alanine (i.e., the major isotype contains 29 alanine residues). Analysis of the major hake isotype by natural abundance carbon-13 NMR spectroscopy has indicated that the protein is remarkably similar in conformation to the homologous protein from carp ( Cyprinus carpio). Carbon-13 NMR spectra obtained on the major hake parvalbumin isotype in which the calcium has been replaced with trivalent ytterbium (Yb(III)) are similar to spectra obtained previously with the carp protein. The present study also provides evidence that the major parvalbumin isotype from the silver hake has the ability to bind the hydrophobic fluorescence probe molecule 1-anilino-naphthalene-8-sulfonate (ANS) in the absence of Ca(II) (or Cd(II)) and in the presence of Mg(II), suggesting the existence of a possible hydrophobic recognition site for other proteins.

Intercalation Complex of Proflavine with DNA: Structure and Dynamics by Solid-State NMR

The structure of the complex formed between the intercalating agent proflavine and fibrous native DNA was studied by one- and two-dimensional high-resolution solid-state nuclear magnetic resonance (NMR). Carbon-13-labeled proflavine was used to show that the drug is stacked with the aromatic ring plane perpendicular to the fiber axis and that it is essentially immobile. Natural abundance carbon-13 NMR of the DNA itself shows that proflavine binding does not change the puckering of the deoxyribose ring. However, phosphorus-31 NMR spectra show profound changes in the orientation of the phosphodiester grouping on proflavine binding, with some of the phosphodiesters tilting almost parallel to the helix axis, and a second set almost perpendicular. The first group to the phosphodiesters probably spans the intercalation sites, whereas the tilting of the second set likely compensates for the unwinding of the DNA by the intercalator.

Multinuclear NMR spectroscopic studies of aryltrimethylsilanes and aryldimethylphosphaneboranes

Proton, boron-11, carbon-13, silicon-29, and phosphorus-31 NMR chemical shifts and coupling constants are reported for nine ortho- and 2,6-disubstituted aryltrimethylsilanes and five similarly substituted aryldimethylphosphaneboranes. Resonances in the natural-abundance carbon-13 NMR spectra for both sets of derivatives are assigned on the basis of additivity relationships, proton-coupled spectra, and relative magnitudes of | J( 31P 13C) | coupling constants. Carbon-13 chemical shifts and | 1 J( 13C 1H) | coupling constants indicate that the P(BH 3)(CH 3) 2 group is electron-withdrawing. The 13C chemical shifts of aryl C(5) carbons can be attributed to steric inhibition of resonance of about the same magnitude as that produced by ortho-Si(CH 3) 3. Chemical shift and coupling constant data from previous work are expanded in terms of Taft's dual substituent constants σ 1 and σ R 0. Least squares solutions of these equations for aryldimethylphosphaneborane derivatives provide values of 0.41 for σ 1 and 0.04 for σ R 0 for the P(BH 3)(CH 3) 2 group. These constants produce reasonable agreement with observed 13C chemical shifts and coupling constants in the ortho derivatives.