Narrowband excitation of (2)H powder pattern and its application to (2)H 1D exchange sample-turning NMR.

Frequency-selective narrowband excitation of the (2)H powder pattern was examined. Selection of a single spectral band with a linewidth of ca. 15 kHz was achieved by a narrowband (1)H --> (2)H cross polarization by using the time-averaged precession frequency method. Further narrowing with a ca. 5 kHz linewidth is achieved by DANTE irradiation. The narrowband excitation was applied to transform a recently developed 2D spin-exchange method for obtaining structural information (Chem. Phys. Lett. 260, 159, (1996)) into its 1D analogue. The determination of the D-C-D bond angle was demonstrated for alpha-glycine-[2,2-d(2)]. Further, the intermolecular polarization transfer between two deuterons separated by 0.299 nm was detected with the mixing time of 500 ms.     

Narrowband Excitation of H Powder Pattern and Its Application to H 1D Exchange Sample-Turning NMR

Frequency-selective narrowband excitation of the ²H powder pattern was examined. Selection of a single spectral band with a linewidth of ca. 15 kHz was achieved by a narrowband ¹H → ²H cross polarization by using the time-averaged precession frequency method. Further narrowing with a ca. 5 kHz linewidth is achieved by DANTE irradiation. The narrowband excitation was applied to transform a recently developed 2D spin-exchange method for obtaining structural information (Chem. Phys. Lett. 260, 159, (1996)) into its 1D analogue. The determination of the D–C–D bond angle was demonstrated for α-glycine-[2,2-d₂]. Further, the intermolecular polarization transfer between two deuterons separated by 0.299 nm was detected with the mixing time of 500 ms.     

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.     

N—甲基异靛蓝在不同溶剂中的^1H—NMR研究

用选择照射方法研究了N-甲基异靛蓝(isoindigatin)在(CD_3)_2CO、CDCl_3、DMSO-d_6溶剂中及重水交换后的~1H NMR谱。得到了化学环境极为类似的两芳环质子在不同溶剂中的NMR谱线归属和偶合常数。     

Superslow Backbone Protein Dynamics as Studied by 1D Solid-State MAS Exchange NMR Spectroscopy

Superslow backbone dynamics of the protein barstar and the polypeptide polyglycine was studied by means of a solid-state MAS 1D exchange NMR method (time-reverse ODESSA) that can detect reorientation of nuclei carrying anisotropic chemical shift tensors. Experiments were performed on carbonyl 13C in polyglycine (natural abundance) and backbone 15N nuclei in uniformly 15N-enriched barstar within a wide range of temperatures in dry and wet powders for both samples. Two exchange processes were observed in the experiments: molecular reorientation and spin diffusion. Experimental conditions that are necessary to separate these two processes are discussed on a quantitative level. It was revealed that the wet protein undergoes molecular motion in the millisecond range of correlation times, whereas in dry protein and polyglycine molecular reorientations could not be detected. The correlation time of the motion in the wet barstar at room temperature is 50-100 ms; the activation energy is about 80 kJ/mol. Previously, protein motions with such a long correlation time could be observed only by methods detecting chemical exchange in solution (e.g., hydrogen exchange). The application of solid-state MAS exchange spectroscopy provides new opportunities in studying slow biomolecular dynamics that is important for the biological function of proteins.     

Effects of various types of molecular dynamics on 1D and 2D (2)H NMR studied by random walk simulations

By carrying out random walk simulations we systematically study the effects of various types of complex molecular dynamics on (2)H NMR experiments in solids. More precisely, we calculate one-dimensional (1D) (2)H NMR spectra and the results of two dimensional (2D) (2)H NMR experiments in time domain, taking into account isotropic as well as highly restricted motions which involve rotational jumps about different finite angles. Although the dynamical models are chosen to mimic the primary and secondary relaxation in supercooled liquids and glasses, we do not intend to describe experimental results quantitatively but rather to show general effects appearing for complex reorientations. We carefully investigate whether 2D (2)H NMR in time domain, which was originally designed to measure correlation times of ultraslow motions (tau >/= 1 ms), can be used to obtain shorter tau, too. It is demonstrated that an extension of the time window to tau >/= 10 &mgr;s is possible when dealing with exponential relaxation, but that it will fail if there is a distribution of correlation times G(lgtau). Vice versa, we show that 1D (2)H NMR spectra, usually recorded to look at dynamics with tau in the microsecond regime, are also applicable for studying ultraslow motions provided that the loss of correlation is achieved step by step. Therefore, it is useful to carry out 1D and 2D NMR experiments simultaneously in order to reveal the mechanism of complex molecular motions. In addition, we demonstrate that highly restricted dynamics can be clearly observed in 1D spectra and in 2D NMR in time domain if long solid-echo delays and large evolution times are applied, respectively. Finally, unexpected observations are described which appear in the latter experiment when considering very broad distributions G(lgtau). Because of these effects, time scale and geometry of a considered motion cannot be extracted from a straightforward analysis of experimental results. Copyright 2000 Academic Press.     

Measurement of the local 1H spin-diffusion coefficient in polymers

Proton spin diffusion is widely used to determine domain sizes in heterogeneous organic solids. For an accurate analysis, spin diffusion coefficients are required. However, in most cases they are not directly measured, but instead derived from model systems. The effects of magic-angle spinning (MAS), mobility, or spin-lock fields on spin-diffusion coefficients have also been difficult to quantify. In this work, direct measurement of local (1)H spin-diffusion coefficients in any rigid polymer is achieved in experiments with heteronuclear dephasing of the (1)H magnetization, a mixing time for (1)H spin diffusion, and (13)C detection after cross-polarization. In the presence of (1)H homonuclear decoupling and (13)C 180 degrees-pulse recoupling, each (13)C spin dephases a significant number (3-20) of protons, depending on the dephasing time. For (13)C and other sufficiently dilute heteronuclei, the dephasing of the protons is described by simple spin-pair REDOR curves. As a result, every (13)C nucleus will "burn" a spherical hole of known diameter and profile into the proton magnetization distribution. (1)H spin diffusion into the hole during the mixing time can be monitored and simulated accurately for every resolved (13)C site, with the spin-diffusion coefficient as the only significant unknown parameter. By varying the dephasing time, holes with diameters of 0.4-0.8 nm can be burned into the proton magnetization profile and thus the dependence of the local spin-diffusion coefficients on the proton density or partial mobility can be explored. The effects of transverse or magic-angle spin-lock fields on spin diffusion can be quantified conveniently by this method. Analytical and numerical fits yield short-range spin-diffusion coefficients of 0.2-0.5 nm(2)/ms on the 0.5-nm scale, which is smaller than the value of 0.8 nm(2)/ms for organic solids previously measured on the 10-nm scale.     

Observing in-phase single-quantum 15N multiplets for NH2/NH3+ groups with two-dimensional heteronuclear correlation spectroscopy

Two-dimensional (2D) F1-(1)H-coupled HSQC experiments provide 3:1:1:3 and 1:0:1 multiplets for AX(3) and AX(2) spin systems, respectively. These multiplets occur because, in addition to the 2S(y)H(z)(a)-->2S(y)H(z)(a) process, the coherence transfers such as 2S(y)H(z)(a)-->2S(y)H(z)(b) occurring in t(1) period provide detectable magnetization during the t(2) period. Here, we present a 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiment that provides a 1:3:3:1 quartet for AX(3) spin system and a 1:2:1 triplet for AX(2). The experiment is a derivative of 2D HISQC experiment [J. Iwahara, Y.S. Jung, G.M. Clore, Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation. J. Am. Chem. Soc. 129 (2007) 2971-2980] and contains a scheme that kills anti-phase single-quantum terms generated in the t(1) period. The purge scheme is essential to observe in-phase single-quantum multiplets. Applications to the NH(2) and NH(3)(+) groups in proteins are demonstrated.     

High-resolution four-dimensional carbon-correlated 1H-1H ROESY experiments employing isotags and the filter diagonalization method for effective assignment of glycosidic linkages in oligosaccharides

Four-dimensional nuclear magnetic resonance spectroscopy of oligosaccharides that correlates 1H-1H ROESY cross peaks to two additional 13C frequency dimensions is reported. The 13C frequencies were introduced by derivatization of all free hydroxyl groups with doubly 13C-labeled acetyl isotags. Pulse sequences were optimized for processing with the filter diagonalization method. The extensive overlap typically observed in 2D ROESY 1H-1H planes was alleviated by resolution of ROESY cross peaks in the two added dimensions associated with the carbon frequencies of the isotags. This enabled the interresidue 1H-1H ROESY cross peaks to be unambiguously assigned hence spatially proximate sugar spin systems across glycosidic bonds could be effectively ascertained. An experiment that selectively amplifies interresidue ROESY 1H-1H cross peaks is also reported. It moves the magnetization of an intraresidue proton normally correlated to a sugar H-1 signal orthogonally along the z axis prior to a Tr-ROESY mixing sequence. This virtually eliminates the incoherent intraresidue ROESY transfer, suppresses coherent TOCSY transfer, and markedly enhances the intensity of interresidue ROESY cross peaks.     

Measurement of one-bond 13Calpha-1Halpha residual dipolar coupling constants in proteins by selective manipulation of CalphaHalpha spins

We have developed new 2D and 3D experiments for the measurement of C(alpha)-H(alpha) residual dipolar coupling constants in (13)C and (15)N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C(alpha)-H(alpha) splitting by means of C(alpha) magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H(alpha) magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the (15)N chemical shifts and the IPAP principle is used to obtain (1)H-(15)N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C(alpha) or H(alpha) nuclei. This was made possible by the use of C(alpha) selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods ( approximately 25 ms) yielding higher precision C(alpha)-H(alpha) splitting determination than is achievable with existing frequency based methods.     

A study of dipolar interactions and dynamic processes of water molecules in tendon by 1H and 2H homonuclear and heteronuclear multiple-quantum-filtered NMR spectroscopy

The effect of proton exchange on the measurement of 1H-1H, 1H-2H, and 2H-2H residual dipolar interactions in water molecules in bovine Achilles tendons was investigated using double-quantum-filtered (DQF) NMR and new pulse sequences based on heteronuclear and homonuclear multiple-quantum filtering (MQF). Derivation of theoretical expressions for these techniques allowed evaluation of the 1H-1H and 1H-2H residual dipolar interactions and the proton exchange rate at a temperature of 24 degrees C and above, where no dipolar splitting is evident. The values obtained for these parameters at 24 degrees C were 300 and 50 Hz and 3000 s-1, respectively. The results for the residual dipolar interactions were verified by repeating the above measurements at a temperature of 1.5 degrees C, where the spectra of the H2O molecules were well resolved, so that the 1H-1H dipolar interaction could be determined directly from the observed splitting. Analysis of the MQF experiments at 1.5 degrees C, where the proton exchange was in the intermediate regime for the 1H-2H dipolar interaction, confirmed the result obtained at 24 degrees C for this interaction. A strong dependence of the intensities of the MQF signals on the proton exchange rate, in the intermediate and the fast exchange regimes, was observed and theoretically interpreted. This leads to the conclusion that the MQF techniques are mostly useful for tissues where the residual dipolar interaction is not significantly smaller than the proton exchange rate. Dependence of the relaxation times and signal intensities of the MQF experiments on the orientation of the tendon with respect to the magnetic field was observed and analyzed. One of the results of the theoretical analysis is that, in the fast exchange regime, the signal decay rates in the MQF experiments as well as in the spin echo or CPMG pulse sequences (T2) depend on the orientation as the square of the second-rank Legendre polynomial.     

Geometry of multiple-spin systems as reflected in 13C-{1H} dipolar spectra measured at Lee-Goldburg cross-polarization

Theoretical calculation and analysis of (13)C-{(1)H} dipolar spectra of small-size spin clusters is presented. Dipolar spectra simulated using the time-independent average Hamiltonian are compared with the dipolar profiles obtained by 2D and 3D (1)H-(13)C correlation experiments employing Lee-Goldburg off-resonance cross-polarization (LG-CP). It is demonstrated that the structural parameters such as interatomic distances as well as mutual orientation of internuclear vectors can be derived from the dipolar profiles of simple spin clusters. Simplified analysis of the dipolar spectra based on isolated-like spin-pair approach can be used only if interacting spin cluster is reduced to the three-spin system in which the angle between both internuclear vectors ranges from 45 degrees to 135 degrees . For other local arrangements of spin systems the produced dipolar spectra must be analyzed with high caution. Contributions of all interacting spins to dipolar evolution of (13)C magnetization are mutually mixed and cannot be easily separated. However, simplification of the dipolar spectra is achieved by selective excitation. Enhanced selectivity of LG-CP transfer due to the initial (1)H chemical-shift-evolution period makes it possible to construct the dipolar spectra from (1)H-(13)C cross-peak intensities for every detected (1)H-(13)C spin-pair. Consequently, isolated-like spin pair evolution of the detected (1)H-(13)C coherence dominates to the resulting dipolar profile, while the influence of other interacting spins is suppressed. However, this suppression is not quite complete and analysis of the selective dipolar spectra based on isolated-like spin-pair approach cannot be used generally. Especially evolution of long-range (1)H-(13)C coherence is still significantly affected by spin states of other coupled hydrogen atoms.     

S(3)E-E.COSY methods for the measurement of (19)F associated scalar and dipolar coupling constants

A (1)H-(19)F spin state selective excitation (S(3)E) pulse sequence element has been applied in combination with (1)H homonuclear mixing to create E.COSY-type experiments designed to measure scalar J(HF2') and J(HH2') and residual dipolar D(HF2') and D(HH2') couplings in 2'-deoxy-2'-fluoro-sugars. The (1)H-(19)F S(3)E pulse sequence element, which resembles a simple INEPT sequence, achieves spin-state-selective correlation between geminal (1)H-(19)F spin pairs by linear combination of in-phase (19)F magnetization and anti-phase magnetization evolved from (1)H. Since the S(3)E sequence converts both (19)F and (1)H steady-state polarization into observable coherences, an approximately twofold signal increase is observed for fully relaxed (1)H-(19)F spin pairs with respect to a standard (1)H coupled (19)F 1D experiment. The improved sensitivity and resolution afforded by the use of (1)H-(19)F S(3)E E.COSY-type experiments for measuring couplings is demonstrated on the nucleoside 9-(2',3'-dideoxy-2'-fluoro-beta-D-threo-pentofuranosyl)adenine (beta-FddA) and on a selectively 2'-fluorine labeled 21mer RNA oligonucleotide.     

Electron spin resonance spectroscopy of molecules in large precessional motion: a case of H6(+) and H4D2(+) in solid parahydrogen

We have measured electron spin resonance (ESR) spectra of H6+ and H4D2(+) ions produced in gamma-ray irradiated solid parahydrogen. Anisotropic hyperfine-coupling constants for H6(+) and H4D2(+) determined by the analysis of ESR lines at 4.2K were -0.06 and -0.12 mT, respectively, which were opposite in sign to and much smaller than theoretical results of 1.17-1.25 mT. Although no change was observed in H6(+), the constant for H4D2(+) increased to be 1.17 mT at 1.7 K, which is very close to the theoretical value. We concluded that H6+ both at 4.2 and 1.7 K and H4D2(+) at 4.2K should be in a large precessional motion with the angle of 57-59 degrees, but the precession of H4D2(+) is stopped at 1.7 K.     

2H2O quadrupolar splitting used to measure water exchange in erythrocytes

The 2H NMR resonance from HDO (D=2H) in human red blood cells (RBCs) suspended in gelatin that was held stretched in a special apparatus was distinct from the two signals that were symmetrically arranged on either side of it, which were assigned to extracellular HDO. The large extracellular splitting is due to the interaction of the electric quadrupole moment of the 2H nuclei with the electric field gradient tensor of the stretched, partially aligned gelatin. Lack of resolved splitting of the intracellular resonance indicated greatly diminished or absent ordering of the HDO inside RBCs. The separate resonances enabled the application of a saturation transfer method to estimate the rate constants of transmembrane exchange of water in RBCs. However both the theory and the practical applications needed modifications because even in the absence of RBCs the HDO resonances were maximally suppressed when the saturating radio-frequency radiation was applied exactly at the central frequency between the two resonances of the quadrupolar HDO doublet. More statistically robust estimates of the exchange rate constants were obtained by applying two-dimensional exchange spectroscopy (2D EXSY), with back-transformation analysis. A monotonic dependence of the estimates of the efflux rate constants on the mixing time, tmix, used in the 2D EXSY experiment were seen. Extrapolation to tmix=0, gave an estimate of the efflux rate constant at 15 degrees C of 31.5+/-2.2 s(-1) while at 25 degrees C it was approximately 50 s(-1). These values are close to, but less than, those estimated by an NMR relaxation-enhancement method that uses Mn2+ doping of the extracellular medium. The basis for this difference is thought to include the high viscosity of the extracellular gel. At the abstract level of quantum mechanics we have used the quadrupolar Hamiltonian to provide chemical shift separation between signals from spin populations across cell membranes; this is the first time, to our knowledge, that this has been achieved.     

Amide proton relaxation measurements employing a highly deuterated protein

Proton NMR longitudinal and transverse relaxation rates of unlabelled proteins are generally dominated by the many 1H-1H dipolar interactions so that spin diffusion, rather than molecular or internal motions, governs longitudinal relaxation. Here, relaxation measurements of backbone amide proton (1H(N)) magnetisations have been carried out employing the 99% 2H, 98% 15N labelled, small 2F2 protein domain in 10%/90% H(2)O/D(2)O solution. Under these conditions, the longitudinal relaxation rates exhibit time constants, T(1)*=1/R(1)* if described by a mono-exponential, within the range of 3.0 to 18.7s-a wide range which indicates that the phenomenon of spin diffusion has been greatly reduced. The majority of 1H(N) nuclei in this sample (pH 4.0 and 5 degrees C) exhibit chemical exchange with solvent that couples their longitudinal relaxation to that of the solvent. For the subset of 1H(N) nuclei not undergoing detectable solvent chemical exchange, the R(1)* rates correlate well with their individual 1H(N,O)/2H(N,O) structural environments. The correlation for corresponding transverse relaxation rates, R(2)* was found to be less good. Longitudinal relaxation measurements in 1%/99% H(2)O/D(2)O solution identify a further subset of 1H(N) nuclei which exhibit essentially indistinguishable R(1)* rates in both 1% and 10% H(2)O, implying that averaging of rates from spin diffusion processes and different 2F2 isotopomer populations are negligible for these 1H(N) sites. In addition to a high sensitivity to structural parameters, model calculations predict 1H(N) relaxation rates to exhibit pronounced sensitivity to internal dynamics.     

(1)H-(13)C INEPT MAS NMR correlation experiments with (1)H-(1)H mediated magnetization exchange to probe organization in lipid biomembranes

Two-dimensional (1)H-(13)C INEPT MAS NMR experiments utilizing a (1)H-(1)H magnetization exchange mixing period are presented for characterization of lipid systems. The introduction of the exchange period allows for structural information to be obtained via (1)H-(1)H dipolar couplings but with (13)C chemical shift resolution. It is shown that utilizing a RFDR recoupling sequence with short mixing times in place of the more standard NOE cross-relaxation for magnetization exchange during the mixing period allowed for the identification and separation of close (1)H-(1)H dipolar contacts versus longer-range inter-molecular (1)H-(1)H dipolar cross-relaxation. These 2D INEPT experiments were used to address both intra- and inter-molecular contacts in lipid and lipid/cholesterol mixtures.     

Anisotropic hyperfine interactions limit the efficiency of spin-exchange optical pumping of ³He nuclei

We use accurate ab initio and quantum scattering calculations to demonstrate that the maximum 3He spin polarization that can be achieved in spin-exchange collisions with potassium (3?K) and silver (1??Ag) atoms is limited by the anisotropic hyperfine interaction. We find that spin exchange in Ag-He collisions occurs much faster than in K-He collisions over a wide range of temperatures (10-600 K). Our analysis indicates that measurements of trap loss rates of 2S atoms in the presence of cold 3He gas may be used to probe anisotropic spin-dependent interactions in atom-He collisions.     

2D 1H-31P solid-state NMR studies of the dependence of inter-bilayer water dynamics on lipid headgroup structure and membrane peptides

The dynamics of hydration-water in several phospholipid membranes of different compositions is studied by 2D (1)H-(31)P heteronuclear correlation NMR under magic-angle spinning. By using a (1)H T(2) filter before and a (1)H mixing-time after the evolution period and (31)P detection, inter-bilayer water is selectively detected without resonance overlap from bulk water outside the multilamellar vesicles. Moreover the (1)H T(2) relaxation time of the inter-bilayer water is measured. Lipid membranes with labile protons either in the lipid headgroup or in sterols exhibit water-(31)P correlation peaks while membranes free of exchangeable protons do not, indicating that the mechanism for water-lipid correlation is chemical exchange followed by relayed magnetization transfer to (31)P. In the absence of membrane proteins, the inter-bilayer water (1)H T(2)'s are several tens of milliseconds. Incorporation of charged membrane peptides shortened this inter-bilayer water T(2) significantly. This T(2) reduction is attributed to the peptides' exchangeable protons, molecular motion and intermolecular hydrogen bonding, which affect the water dynamics and the chemically relayed magnetization transfer process.     

精确测量慢魔角旋转条件下碳氢偶极谱

发展了精确测量二维固体魔角旋转条件下碳氢偶极谱的方法，用以分析固体高分子分子运动，并给出了全同立构聚丙烯的实验谱.