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1.
We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive 13C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective 13C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-13C]glucose preferentially labels the ends of the side chains, while [2-13C]glycerol labels the Cα of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively 13C labeled protein were performed using 15N–13C 2D correlation spectroscopy. From the time dependence of the 15N–13C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective 13C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state. 相似文献
2.
Katarzyna Paradowska Micha Wolniak Maciej Pisklak Jan A. Gliski Matthew H. Davey Iwona Wawer 《Solid state nuclear magnetic resonance》2008,34(4):202-209
Oxindole alkaloids, isolated from the bark of Uncaria tomentosa [Willd. ex Schult.] Rubiaceae, are considered to be responsible for the biological activity of this herb. Five pentacyclic and two tetracyclic alkaloids were studied by solid-state NMR and theoretical GIAO DFT methods. The 13C and 15N CPMAS NMR spectra were recorded for mitraphylline, isomitraphylline, pteropodine (uncarine C), isopteropodine (uncarine E), speciophylline (uncarine D), rhynchophylline and isorhynchophylline. Theoretical GIAO DFT calculations of shielding constants provide arguments for identification of asymmetric centers and proper assignment of NMR spectra. These alkaloids are 7R/7S and 20R/20S stereoisomeric pairs. Based on the 13C CP MAS chemical shifts the 7S alkaloids (δ C3 70–71 ppm) can be easily and conveniently distinguished from 7R (δC3 74.5–74.9 ppm), also 20R (δC20 41.3–41.7 ppm) from the 20S (δC20 36.3–38.3 ppm). The epiallo-type isomer (3R, 20S) of speciophylline is characterized by a larger 15N MAS chemical shift of N4 (64.6 ppm) than the allo-type (3S, 20S) of isopteropodine (δN4 53.3 ppm). 15N MAS chemical shifts of N1–H in pentacyclic alkaloids are within 131.9–140.4 ppm. 相似文献
3.
Mansoureh Mousavi Steve S.-F. Yu Der-Lii M. Tzou 《Solid state nuclear magnetic resonance》2009,36(1):24-31
13C cross-polarization/magic-angle spinning (CP/MAS) solid-state NMR spectroscopy has been employed to analyze four vitamin D compounds, namely vitamin D3 (D3), vitamin D2 (D2), and the precursors ergosterol (Erg) and 7-dehydrocholesterol (7DHC). The 13C NMR spectrum of D3 displays a doublet pattern for each of the carbon atoms, while that of Erg contains both singlet and doublet patterns. In the cases of 7DHC and D2, the 13C spectra display various multiplet patterns, viz. singlets, doublets, triplets, and quartets. To overcome the signal overlap between the 13C resonances of protonated and unprotonated carbons, we have subjected these vitamin D compounds to 1D 1H-filtered 13C CP/MAS and {1H}/13C heteronuclear correlation (Hetcor) NMR experiments. As a result, assisted by solution NMR data, all of the 13C resonances have been successfully assigned to the respective carbon atoms of these vitamin D compounds. The 13C multiplets are interpreted due to the presence of s-cis and s-trans configurations in the α- and β-molecular conformers, consistent with computer molecular modeling determined by molecular dynamics and energy minimization calculations. To further characterize the ring conformations in D3, we have successfully extracted chemical shift tensor elements for the 13C doublets. It is demonstrated that 13C solid-state NMR spectroscopy provides a robust and high sensitive means of characterizing molecular conformations in vitamin D compounds. 相似文献
4.
Jutta Pauli Barth van Rossum Hans Frster Huub J. M. de Groot Hartmut Oschkinat 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2000,143(2):411
Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid α-spectrin SH3 domain were generated in four different ways, and their 13C CPMAS spectra were compared. The spectrum of a [u-13C, 15N]-labeled sample generated by precipitation shows very narrow 13C signals and resolved scalar carbon–carbon couplings. Linewidths of 16–19 Hz were found for the three alanine Cβ signals of a selectively labeled [70% 3-13C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D 13C–13C RFDR spectra of the [u-13C,15N]-labeled SH3 sample. A comparison of the 13C chemical shifts of the found signal patterns with the 13C assignment obtained in solution shows an intriguing match. 相似文献
5.
Zhe Zhou Rainer Kümmerle James C. Stevens David Redwine Yiyong He Xiaohua Qiu Rongjuan Cong Jerzy Klosin Nikki Montaez Gordon Roof 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2009,200(2):328-333
Recently, a high temperature 10 mm cryoprobe was developed. This probe provides a significant sensitivity enhancement for 13C NMR of polyolefins at a sample temperature of 120–135 °C, as compared to conventional probes. This greatly increases the speed of NMR studies of comonomer content, sequence distribution, stereo- and regioerrors, saturated chain end, unsaturation, and diffusion of polymers. In this contribution, we first compare the 13C NMR sensitivity of this probe with conventional probes. Then, we demonstrate one of the advantages of this probe in its ability to perform 2D Incredible Natural Abundance Double Quantum Transfer Experiment (2D INADEQUATE) in a relatively short period of time. The 2D INADEQUATE has been rarely used for polymer studies because of its inherently very low sensitivity. It becomes even more challenging for studying infrequent polyolefin microstructures, as low probability microstructures represent a small fraction of carbons in the sample. Here, the 2D INADEQUATE experiment was used to assign the 13C NMR peaks of 2,1-insertion regioerrors in a poly(propylene-co-1-octene) copolymer. 相似文献
6.
Ravi Kc Ian D. Henry Gregory H.J. Park Daniel Raftery 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2009,197(2):186-192
Improved NMR detection of mass limited samples can be obtained by taking advantage of the mass sensitivity of microcoil NMR, while throughput issues can be addressed using multiple, parallel sample detection coils. We present the design and construction of a double resonance 300-MHz dual volume microcoil NMR probe with thermally etched 440-nL detection volumes and fused silica transfer lines for high-throughput stopped-flow or flow-through sample analysis. Two orthogonal solenoidal detection coils and the novel use of shielded inductors allowed the construction of a probe with negligible radio-frequency cross talk. The probe was resonated at 1H–2D (upper coil) and 1H–13C (lower coil) frequencies such that it could perform 1D and 2D experiments with active locking frequency. The coils exhibited line widths of 0.8–1.1 Hz with good mass sensitivity for both 1H and 13C NMR detection. 13C-directly detected 2D HETCOR spectra of 5% v/v 13C labeled acetic acid were obtained in less than 5 min. Demonstration of the probe characteristics as well as applications of the versatile two-coil double resonance probe are discussed. 相似文献
7.
Siegel R Trébosc J Amoureux JP Gan Z 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2008,193(2):321-325
Nitrogen-14 (spin I = 1) has always been a nucleus difficult to observe in solid-state NMR and until recently its observation was restricted to one-dimensional (1D) spectra. We present here the first 3D 1H–13C–14N NMR correlation spectrum. This spectrum was acquired on a test sample l-histidine·HCl·H2O using a recently developed technique, which consists in indirectly observing 14N nuclei via dipolar recoupling with an HMQC-type experiment. 相似文献
8.
Louise Odgaard Mads Bak Hans J. Jakobsen Niels Chr. Nielsen 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2001,148(2):298
This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of 13C chemical shift tensors relative to one or more 13C–14N internuclear axes from 13C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic 13C chemical shift and 13C–14N dipole–dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the 13C spins only. The method is demonstrated by experiments and numerical simulations for the 13Cα spins in powder samples of
-alanine and glycine with 13C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential 14N spins in a peptide chain (14Ni–13Cαi–14Ni+1 and 14Ni+1–13C′i–14Ni three-spin systems) as well as residual quadrupolar–dipolar coupling cross-terms is analyzed numerically. 相似文献
9.
A recently proposed 13C–1H recoupling sequence operative under fast magic-angle spinning (MAS) [K. Takegoshi, T. Terao, Solid State Nucl. Magn. Reson. 13 (1999) 203–212.] is applied to observe 13C–1H and 15N–1H dipolar powder patterns in the 1H–15N–13C–1H system of a peptide bond. Both patterns are correlated by 15N-to-13C cross polarization to observe one- or two-dimensional (1D or 2D) correlation spectra, which can be simulated by using a simple analytical expression to determine the H–N–C–H dihedral angle. The 1D and 2D experiments were applied to N-acetyl[1,2-13C,15N]
-valine, and the peptide φ angle was determined with high precision by the 2D experiment to be ±155.0°±1.2°. The positive one is in good agreement with the X-ray value of 154°±5°. The 1D experiment provided the value of φ=±156.0°±0.8°. 相似文献
10.
Gang Zheng Allan M. Torres William S. Price 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2009,198(2):271-274
A new multi-quantum stimulated echo based pulsed gradient spin-echo sequence, MQ-PGSTE, has been developed for measuring translational diffusion. The new sequence provides a higher signal-to-noise ratio than the (Hahn spin-echo based) MAXY-D sequence at long diffusion times, and thus potentially affords better diffusion measurements on macromolecule samples. Based on multi-quantum coherence encoding, the MQ-PGSTE sequence needs considerably lower gradient strengths for diffusion characterization compared to standard single quantum pulsed gradient spin-echo sequences. By using the new sequence, the diffusion coefficient of l-[3-13C]-alanine was found to be 8.1 ± 0.1 × 10−10 m2 s−1, which is in line with the value obtained by the use of the standard stimulated echo based pulsed gradient spin-echo sequence. 相似文献
11.
Eric B. Brouwer Robin Challoner Robin K. Harris 《Solid state nuclear magnetic resonance》2000,18(1-4)
The supramolecular 1 : 1 host–guest inclusion compound, p-tert-butylcalix[4]arene ·α,α,α-trifluorotoluene, 1, is characterized by 19F and 13C solid-state NMR spectroscopy. Whereas the 13C NMR spectra are easily interpreted in the context of earlier work on similar host–guest compounds, the 19F NMR spectra of solid 1 are, initially, more difficult to understand. The 19F{1H} NMR spectrum obtained under cross-polarization and magic-angle spinning conditions shows a single isotropic resonance with a significant spinning sideband manifold. The static 19F{1H} CP NMR spectrum consists of a powder pattern dominated by the contributions of the anisotropic chemical shift and the homonuclear dipolar interactions. The 19F MREV-8 experiment, which minimizes the 19F–19F dipolar contribution, helps to identify the chemical shift contribution as an axial lineshape. The full static 19F{1H} CP NMR spectrum is analysed using subspectral analysis and subsequently simulated as a function of the 19F–19F internuclear distance (DFF = 2.25 ± 0.01 Å) of the rapidly rotating CF3 group without including contributions from additional libration motions and the anisotropy in the scalar tensor. The shielding span is found to be 56 ppm. The width of the centerband in the 19F{1H} sample-spinning CP NMR spectrum is very sensitive to the angle between the rotor and the magnetic field. Compound 1 is thus an attractive standard for setting the magic angle for NMR probes containing a fluorine channel with a proton-decoupling facility. 相似文献
12.
Wiench JW Lin VS Pruski M 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2008,193(2):233-242
A remarkable enhancement of sensitivity can be often achieved in 29Si solid-state NMR by applying the well-known Carr–Purcell–Meiboom–Gill (CPMG) train of rotor-synchronized π pulses during the detection of silicon magnetization. Here, several one- and two-dimensional (1D and 2D) techniques are used to demonstrate the capabilities of this approach. Examples include 1D 29Si{X} CPMAS spectra and 2D 29Si{X} HETCOR spectra of mesoporous silicas, zeolites and minerals, where X = 1H or 27Al. Data processing methods, experimental strategies and sensitivity limits are discussed and illustrated by experiments. The mechanisms of transverse dephasing of 29Si nuclei in solids are analyzed. Fast magic angle spinning, at rates between 25 and 40 kHz, is instrumental in achieving the highest sensitivity gain in some of these experiments. In the case of 29Si–29Si double-quantum techniques, CPMG detection can be exploited to measure homonuclear J-couplings. 相似文献
13.
Thomas Szyperski Csar Fernndez Akira Ono Kurt Wüthrich Masatsune Kainosho 《Journal of magnetic resonance (San Diego, Calif. : 1997)》1999,140(2):491
A two-dimensional {31P} spin-echo-difference constant-time [13C, 1H]-HMQC experiment (2D {31P}-sedct-[13C, 1H]-HMQC) is introduced for measurements of 3JC4′P and 3JH3′P scalar couplings in large 13C-labeled nucleic acids and in DNA–protein complexes. This experiment makes use of the fact that 1H–13C multiple-quantum coherences in macromolecules relax more slowly than the corresponding 13C single-quantum coherences. 3JC4′P and 3JH3′P are related via Karplus-type functions with the phosphodiester torsion angles β and ε, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly 13C, 15N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein–DNA complex. 相似文献
14.
J.L. Crisp S.E. Dann M. Edgar C.G. Blatchford 《Solid state nuclear magnetic resonance》2010,37(3-4):75-81
The polymorphic forms of lactose in alcoholic suspensions have been determined by 13C CP-MAS NMR spectroscopy, employing hand-made glass inserts. Suspensions of alpha lactose monohydrate (Lα·H2O) with particle size between 2 and 200 μm were prepared by 24 h reflux or by storage for 28 d in anhydrous ethanol without agitation. These suspensions were compared to an ethanolic sub-micron lactose suspension provided by a 3 M Health Care (Loughborough). The 13C CP-MAS NMR spectra indicated that Lα·H2O dehydrated to stable anhydrous alpha lactose polymorph (LαS) whilst suspended in ethanol. In addition, strong ethanol 13C resonances were observed for some samples, indicating a liquid–solid interaction between the ethanol and lactose surface. Replacement of ethanol with anhydrous methanol, n-butanol and 3-methylbutan-2-ol implied that the solvent mediated dehydration of Lα·H2O to LαS occurs as a result of sterically controlled interactions. 相似文献
15.
Keiko Kanamori Brian D. Ross 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2001,153(2):193
[5-13C,15N]Glutamine, with 1J(13C–15N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by 13C MRS at 4.7 T. The brain [5-13C]glutamine peak consisted of the doublet from [5-13C,15N]glutamine and the center [5-13C,14N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-13C,15N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-13C,15N]glutamine was formed by glial uptake of released neurotransmitter [5-13C]glutamate and its reaction with 15NH3 catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively13C-enriched by intravenous [2,5-13C]glucose infusion to 13C-label whole-brain glutamate C5, followed by [12C]glucose infusion to chase 13C from the small and rapidly turning-over glial glutamate pool, leaving 13C mainly in the neurotransmitter [5-13C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-13C,15N]glutamine arises from a coupling between 13C of neuronal origin and 15N of glial origin. Measurement of the rate of brain [5-13C,15N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity. 相似文献
16.
The reactions of fullerene[C60] with 2′-azidoethyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (2a) and 2′-azidoethyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside (2b) under ultrasonic irradiation cause the cycloaddition of 2′-azidoethyl glycosides to fullerene[C60] and lead to d-glycosyl fullerene[C60] derivatives 3a and 3b, respectively. The glycosyl fullerene[C60] derivatives were characterized by 1H and 13C NMR, UV–vis, FAB-MS, FT-IR spectra and were a 1:1 glycoside fullerene [C60]-adduct. 相似文献
17.
Fast acquisition of multi-dimensional spectra in solid-state NMR enabled by ultra-fast MAS 总被引:1,自引:1,他引:0
Sgolne Laage Joseph R. Sachleben Stefan Steuernagel Roberta Pierattelli Guido Pintacuda Lyndon Emsley 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2009,196(2):133-141
The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored.It is shown that optimal conditions for performing solid-state 13C NMR under 60 kHz MAS are obtained with low-power CW 1H decoupling, as well as after a low-power 1H,13C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet–van Vleck theory.As a result, optimal 13C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short 1H T1s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D 13C–13C correlation could be acquired in about 24 h on less than 1 mg of SOD sample. 相似文献
18.
V. P. Tarasov Yu. B. Muravlev V. N. Fokin Yu. M Shul'ga 《Journal of Applied Spectroscopy》2003,70(2):315-318
Deuterofullerites C60D
x
have been studied by means of 2H NMR spectroscopy. It has been established that there are two types of carbon–deuterium bindings in the samples under study: tip C–D with quadrupole constant coupling (QCC) 171 kHz and bridge –C...D...C– with QCC 56 kHz. It is possible that the latter bond is a result of the rigidity of the lattice, which is unusual for fullerene compounds. 相似文献
19.
Mihaela Aluas Carmen Tripon John M. Griffin Xenia Filip Vladimir Ladizhansky Robert G. Griffin Steven P. Brown Claudiu Filip 《Journal of magnetic resonance (San Diego, Calif. : 1997)》2009,199(2):173-187
A protocol is presented for correcting the effect of non-specific cross-polarization in CHHC solid-state MAS NMR experiments, thus allowing the recovery of the 1H–1H magnetization exchange functions from the mixing-time dependent buildup of experimental CHHC peak intensity. The presented protocol also incorporates a scaling procedure to take into account the effect of multiplicity of a CH2 or CH3 moiety. Experimental CHHC buildup curves are presented for l-tyrosine·HCl samples where either all or only one in 10 molecules are U–13C labeled. Good agreement between experiment and 11-spin SPINEVOLUTION simulation (including only isotropic 1H chemical shifts) is demonstrated for the initial buildup (tmix < 100 μs) of CHHC peak intensity corresponding to an intramolecular close (2.5 Å) H–H proximity. Differences in the initial CHHC buildup are observed between the one in 10 dilute and 100% samples for cases where there is a close intermolecular H–H proximity in addition to a close intramolecular H–H proximity. For the dilute sample, CHHC cross-peak intensities tended to significantly lower values for long mixing times (500 μs) as compared to the 100% sample. This difference is explained as being due to the dependence of the limiting total magnetization on the ratio Nobs/Ntot between the number of protons that are directly attached to a 13C nucleus and hence contribute significantly to the observed 13C CHHC NMR signal, and the total number of 1H spins into the system. 1H–1H magnetization exchange curves extracted from CHHC spectra for the 100% l-tyrosine·HCl sample exhibit a clear sensitivity to the root sum squared dipolar coupling, with fast buildup being observed for the shortest intramolecular distances (2.5 Å) and slower, yet observable buildup for the longer intermolecular distances (up to 5 Å). 相似文献
20.
A magic-angle spinning (MAS) probe has been constructed which allows the sample to be cooled with helium, while the MAS bearing and drive gases are nitrogen. The sample can be cooled to 25 K using roughly 3 L/h of liquid helium, while the 4-mm diameter rotor spins at 6.7 kHz with good stability (±5 Hz) for many hours. Proton decoupling fields up to at least 130 kHz can be applied. This helium-cooled MAS probe enables a variety of one-dimensional and two-dimensional NMR experiments on biomolecular solids and other materials at low temperatures, with signal-to-noise proportional to 1/T. We show examples of low-temperature 13C NMR data for two biomolecular samples, namely the peptide Aβ14–23 in the form of amyloid fibrils and the protein HP35 in frozen glycerol/water solution. Issues related to temperature calibration, spin–lattice relaxation at low temperatures, paramagnetic doping of frozen solutions, and 13C MAS NMR linewidths are discussed. 相似文献