Use of a water flip-back pulse in the homonuclear NOESY experiment

Summary A simple modification to the WATERGATE water suppression scheme [Piotto, M., Saudek, V. and Sklená, V. (1992) J. Biomol. NMR, 2, 661–665] is proposed. Radiation damping is used as an active element during the mixing time of a NOESY experiment, in order to obtain a reproducable state of the water magnetization at the end of the mixing time. Through the use of a water flip-back pulse and a gradient-tailored excitation scheme, we obtain both an excellent water suppression and a water magnetization close to equilibrium at the beginning of the acquisition time. We show experimentally that this modification results in a 20% gain in intensity for all signals when using a relaxation delay of 1.5 s, and also that avoiding a semisaturated state for the water magnetization allows the amide protons as well as other proton resonances to relax to equilibrium with their proper relaxation time.     

2D selective-TOCSY-DQFCOSY and HSQC-TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide

The assignment of ¹H and ¹³C NMR signals of a complex type triantennary asialooligosaccharide was examined using 2D selective-TOCSY–DQFCOSY and HSQC–TOCSY experiments. The 2D selective-TOCSY–DQFCOSY experiment exhibits a 2D DQFCOSY spectrum of an individual monosaccharide in the undecasaccharide, although the NMR signals of several monosaccharides in the triantennary undecasaccharide are heavily overlapped. Selective excitation of each anomeric proton signal and subsequent TOCSY experiment afforded transverse magnetization corresponding to all of the proton signals of the monosaccharide. This magnetization was then developed with the corresponding DQFCOSY pulse sequence to afford the DQFCOSY spectrum of the individual monosugars. In this case, four GlcNAc-b, -e, -j, and -h residues were excited as a mixture. In order to assign ¹³C signals, a conventional 2D HSQC–TOCSY spectrum was examined and compared with an unambiguous assignment of 2D selective-TOCSY–DQFCOSY thus obtained. This systematic analysis made it possible to obtain an assignment of the ¹H and ¹³C NMR signals of the triantennary undecasaccharide. In addition, these experiments also revealed all of the glycosyl positions in the triantennary undecasaccharide.     

Minimisation of sensitivity losses due to the use of gradient pulses in triple-resonance NMR of proteins

Summary The use of pulsed field gradients in multiple-pulse NMR experiments has many advantages, including the possibility of obtaining excellent water suppression without the need for selective presaturation. In such gradient experiments the water magnetization is dephased deliberately; exchange between the saturated protons of the solvent water and the NH protons of a protein transfers this saturation to the protein. As the solvent is in large excess and relaxes relatively slowly, the result is a reduction in the sensitivity of the experiment due to the fact that the NH proton magnetization is only partially recovered. These effects can be avoided by ensuring that the water magnetization remains intact and is returned to the +z-axis at the start of data acquisition. General procedures for achieving this aim in any triple-resonance experiment are outlined and two specific examples are given. Experimental results confirm the sensitivity advantage of the modified sequences.     

Improving solvent suppression in jump-return NOESY experiments

Summary The problems associated with solvent suppression in jump-return NOESY spectra and in particular the difficulties experienced with using short mixing times are examined. It is shown that the degree of water suppression depends critically on the extent of radiation damping of the water magnetisation during the mixing time of the NOESY sequence. A new jump-return NOESY sequence is proposed which incorporates field gradients and which achieves good levels of water suppression for all values of the mixing time, and for all increments of the NOESY experiment.     

Ribonuclease A--a complete proton NMR fingerprint using TOCSY and NOESY experiments in water

In our attempts to obtain total sequential assignment of the 1H NMR spectrum of ribonuclease A, several published water suppression techniques were tested and assessed. The jump- and-return sequence and its echo hybrid were used with considerable success in both the TOCSY and NOESY experiments on 3mM ribonuclease A solutions. The NMR approach used here may be of general applicability for 1H NMR studies of proteins in water of concentrations under 5mM.     

A 15n-filtered 2D 1H TOCSY experiment for assignment of aromatic ring resonances and selective identification of tyrosine ring resonances in proteins: Description and application to Photoactive Yellow Protein

A new method to selectively detect the ring resonances of the aromatic residues in 15N-labelled proteins is presented. The experiment consists of a 2D 1H TOCSY sequence withremoval of the amide signals via 15N-filtering. Experiments are acquired in the absence andpresence of water inversion; combining the two spectra allows selective observation of thetyrosine ring resonances and enables the identification of their δ andε ring protons. The experiment is demonstrated on a 15N-labelled sample of Photoactive Yellow Protein and isshown to give good selectivity for tyrosine ring resonances under a wide range oftemperatures and pH values.     

SWET for Secure Water Suppression on Probes with High Quality Factor

Water suppression by selective preirradiation is increasingly difficult to achieve on probeheads with high quality factor because of the opposing forces of radiation damping. Here we show that a simple modification to the WET scheme provides reliable water suppression in aqueous solutions of proteins and peptides with minimal saturation of the H^α protons. The scheme is shown to work also with dilute peptide solutions. It is recommended to maintain the water suppression during the evolution time of COSY experiments by weak selective irradiation that causes only minimal Bloch-Siegert shifts. The new water-suppression scheme suppresses the water magnetization by spatial scrambling. Traditional water suppression by preirradiation is similarly based more on water scrambling due to the radiofrequency inhomogeneity than on relaxation effects.Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Summary The backbone ¹H and ¹⁵N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the ¹⁵N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D two-, three-dimensional - drkN SH3 N-terminal SH3 domain of Drosophila drk - HSQC heteronuclear single-quantum spectroscopy - NOE nuclear Overhauser enhancement - SH3 Src homology domain 3 - TOCSY total correlation spectroscopy     

Use of optimized 1D TOCSY NMR for improved quantitation and metabolomic analysis of biofluids

One dimensional selective TOCSY experiments have been shown to be advantageous in providing improved data inputs for principle component analysis (PCA) (Sandusky and Raftery 2005a, b). Better subpopulation cluster resolution in the observed scores plots results from the ability to isolate metabolite signals of interest via the TOCSY based filtering approach. This report reexamines the quantitative aspects of this approach, first by optimizing the 1D TOCSY experiment as it relates to the measurement of biofluid constituent concentrations, and second by comparing the integration of 1D TOCSY read peaks to the bucket integration of 1D proton NMR spectra in terms of precision and accuracy. This comparison indicates that, because of the extensive peak overlap that occurs in the 1D proton NMR spectra of biofluid samples, bucket integrals are often far less accurate as measures of individual constituent concentrations than 1D TOCSY read peaks. Even spectral fitting approaches have proven difficult in the analysis of significantly overlapped spectral regions. Measurements of endogenous taurine made over a sample population of human urine demonstrates that, due to background signals from other constituents, bucket integrals of 1D proton spectra routinely overestimate the taurine concentrations and distort its variation over the sample population. As a result, PCA calculations performed using data matrices incorporating 1D TOCSY determined taurine concentrations produce better scores plot subpopulation cluster resolution.     

Improved residual water suppression: WET180

Water suppression in biological NMR is frequently made inefficient by the presence of faraway water that is located near the edges of the RF coil and experiences significantly reduced RF field. WET180 (WET with 180 degrees pulse-toggling) is proposed to cancel the faraway water contribution to the residual solvent signal. The pulse sequence incorporates a modification of the last WET selective pulse to accommodate insertion of a toggled 180 degrees inversion pulse so that the original WET selective pulse angles are effectively preserved. Compared with existing WET methods, WET180 has the advantages of easy implementation, improved residual water suppression, clean spectral phase properties, and good signal intensity retention. WET180 is expected to be most useful in observing resonances close to water in samples containing biological molecules. In addition, the principle of WET180 can be applied in multidimensional experiments to improve residual water suppression and reduce artifacts around water.     

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accurate Quantitation of Water-amide Proton Exchange Rates Using the Phase-Modulated CLEAN Chemical EXchange (CLEANEX-PM) Approach with a Fast-HSQC (FHSQC) Detection Scheme

Measurement of exchange rates between water and NH protons by magnetization transfer methods is often complicated by artifacts, such as intramolecular NOEs, and/or TOCSY transfer from C protons coincident with the water frequency, or exchange-relayed NOEs from fast exchanging hydroxyl or amine protons. By applying the Phase-Modulated CLEAN chemical EXchange (CLEANEX-PM) spin-locking sequence, 135°(x) 120°(-x) 110°(x) 110°(-x) 120°(x) 135°(-x) during the mixing period, these artifacts can be eliminated, revealing an unambiguous water-NH exchange spectrum. In this paper, the CLEANEX-PM mixing scheme is combined with Fast-HSQC (FHSQC) detection and used to obtain accurate chemical exchange rates from the initial slope analysis for a sample of 15N labeled staphylococcal nuclease. The results are compared to rates obtained using Water EXchange filter (WEX) II-FHSQC, and spin-echo-filtered WEX II-FHSQC measurements, and clearly identify the spurious NOE contributions in the exchange system.     

抗病促生复合芽孢杆菌水分散粒剂的研制与应用

【背景】乳油、可湿性粉剂和粉剂等生物制剂含有苯类有机溶剂及粉尘,会对环境造成污染,而水分散粒剂具有环境友好性、附加值高、市场潜力大等优点,被认为是最具发展前景的剂型之一,然而关于复合芽孢杆菌水分散颗粒的研究却很少。【目的】利用解淀粉芽孢杆菌BZ6-1和短小芽孢杆菌SC-12研制成一种复合芽孢杆菌水分散粒剂。【方法】通过生物相容性实验,研究不同载体和助剂对两种芽孢杆菌孢子的影响,以筛选出最佳载体和添加剂。通过质量检测实验,研究不同筛孔直径、干燥温度、干燥时间对水分散性颗粒质量的影响,以优化制粒条件。在辣椒定植后,使用不同剂量的水分散粒剂进行田间辣椒试验。【结果】复合芽孢杆菌水分散粒剂最佳配方:润湿剂4%十二烷基硫酸钠,分散剂6%羧甲基纤维素钠,崩解剂4%硫酸铵,粘结剂4%聚乙二醇,载体82%硅藻土;最佳造粒条件为:筛孔粒径0.8 mm,烘干温度40°C,烘干时间45 min。研制出的复合芽孢杆菌水分散粒剂含菌量为2.52×108CFU/g,悬浮率为79.3%,pH6.8,水分含量为4.5%,湿润时间为19.6 s,崩解时间为86.4 s,颗粒强度适中,符合水分散粒剂国家标准。【结论】研制...     

Quantitative covariance NMR by regularization

The square root of a covariance spectrum, which offers high spectral resolution along both dimensions requiring only few t ₁ increments, yields in good approximation the idealized 2D FT spectrum provided that the amount of magnetization exchanged between spins is relatively small. When this condition is violated, 2D FT and covariance peak volumes may differ. A regularization method is presented that produces a modified covariance spectrum with cross-peak volumes that closely match their 2D FT analogues. The method is demonstrated for TOCSY spectra with variable mixing times.     

Novel strategies for sensitivity enhancement in heteronuclear multi—dimensional NMR experiments employing pulsed field gradients

Summary Novel strategies for sensitivity enhancement in heteronuclear multidimensional spectra are introduced and evaluated theoretically and experimentally. It is shown that in 3D sequences employing several Coherence Order Selective Coherence Transfer (COS-CT) steps, enhancement factors of up to 2 can be achieved. This sensitivity enhancement is compatible with the use of heteronuclear gradient echoes, yielding spectra with excellent water suppression. HNCO and HCCH-TOCSY pulse sequences are proposed and experimentally tested. These experiments employ recently developed coherence order selective pulse sequence elements, e.g., COS-INEPT and planar TOCSY for antiphase to in-phase transfers 2F^-S₂S^- or in-phaseaCOS-CT for in-phase transfer F^-S^-, and the well-known isotropic TOCSY mixing sequences for homo- and heteronuclear in-phase transfer.This work has been presented in part at the 35th ENC, April 10–15, 1994, Asilomar, CA, U.S.A.     

NMR study of seed hydration with deuterated water: dependence of proton signals on hydration level.

Proton NMR signals in seeds are shown to depend on hydration level. In fact at low water amount, as it occurs in many native seeds, protons can have a restricted mobility and are not detectable. A NMR method for measuring the dependence of proton signals on hydration is reported. The method also allows the separation of the contributions of water and non-water protons in a low-resolution NMR experiment. It is based on successive hydrations (with deuterated water) - desiccation steps and on the analysis of the transverse magnetization decay curves.     

Amplification of radiation damping in a 600-MHz NMR spectrometer: Application to the study of water-protein interactions

Summary A new application of a recently developed electronic radiation-damping (RD) control system is presented. It is possible to amplify radiation damping so as to make the water magnetization return back to its equilibrium direction in a time shorter than the characteristic RD time. Certain types of experiments involving radiation damping as a selective inversion pulse can be significantly improved by this new method. Moreover, amplification of RD is shown to improve water suppression and consequently the dynamics of 2D NOESY experiments on proteins.     

节水农业及其生理生态基础

提高自然降水和灌溉水利用效率是节水农业要解决的中心问题。近年实践证明,通过提高水分利用率的途径增加农田生产力存在很大潜力,节水和增产的目标可能同时实现。为实现这一目标,需要研究确定植物水分亏缺的允许程度。植物各个生理过程对水分亏缺的敏感性不同,综合文献报道和作者研究结果,水分亏缺对与作物产量密切相关生理过程影响的先后顺序为:生长—蒸腾—光合—运输。在一定条件下,有限水分亏缺不会对作物最终经济产量造成影响,但却能显著提高水分利用效率。     

Through-bond correlation of imino and aromatic resonances in 13C-,15N-labeled RNA via heteronuclear TOCSY

Summary Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly ¹⁵N-, ¹³C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), of as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.To whom correspondence should be addressed.