Beryllium fluoride exchange rate accelerated by Mg²âº as discovered by ¹9F NMR.

Beryllium fluoride is widely used as a phosphoryl analogue in macromolecular studies, which are not only fluoride-sensitive but also magnesium-dependent. The beryllium fluorides are a mixture of different species including BeF3(-) and BeF4(2-) exchanging under thermodynamic equilibrium in neutral aqueous solutions. In the cases of mimicking phosphate group transfer, both beryllium fluoride and the magnesium ion are generally needed. However, the impact of magnesium on the bioactivity of beryllium fluoride is not clear. We have found by (19)F NMR spectroscopy that Mg(2+) can severely affect the chemical exchange kinetics between BeF3(-) and BeF4(2-). When the F(-) concentration is relatively low, the presence of 10.0 mM Mg(2+) can accelerate the exchange rate 3-4 fold. However, when the F(-) concentration is relatively high, the Mg(2+) effect on the chemical exchange vanishes. On the basis of these findings, we proposed a possible mechanism that BeF4(2-) and Mg(2+) form an ion pair that affects the distribution of beryllium fluoride species and thus the activity in the solution.

NASR: an effective approach for simultaneous noise and artifact suppression in NMR spectroscopy.

As a powerful tool for biological analysis, especially protein structure and dynamic studies, nuclear magnetic resonance (NMR) spectroscopy suffers from intrinsic low signal to nose ratio (SNR) and long acquisition time required for multidimensional (nD) experiments. Nonuniform sampling (NUS) can effectively speed up the experiment but often introduces artifacts into the spectrum. In addition to the development of highly sensitive hardware and NMR pulse sequences, data postprocessing is a relative simple and cost-effective method to improve the SNR and suppress the artifacts. In this work, we propose an effective approach for simultaneously suppressing noise and artifacts based on the resampling principle. The method is named NASR for short and tested using one-, two-, and three-dimensional (1D, 2D, and 3D) NMR spectra that were acquired using ether conventional or NUS (spiral and random, for 3D) approaches. The results reveal that the NASR is fast and applicable for improving the quality of 1D to nD NMR spectra with all kinds of sampling schemes.

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase.

Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein-protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295-304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many (1)H(N), (13)C, and (15)N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N-H correlations in the (1)H-(15)N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77-83 and residues 127-131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.

(1)H- (31)P soft-HSQC pulse sequence specifically for detecting phosphomono- and diesters in biological samples.

PURPOSE: Phosphomono- and diesters (PME and PDE) are important metabolites that are potential biomarkers for a number of cancers. We designed a new NMR pulse sequence, i.e., (1)H-(31)P soft-heteronuclear single quantum correlation (HSQC), specifically for noninvasively detecting PME and PDE in biological samples. PROCEDURE: The nonselective (1)H refocusing π pulses in the conventional heteronuclear single quantum correlation pulse sequence are replaced by selective π pulses. When the selective pulses are offset on the CH2O resonances, the homonuclear couplings between the NCH2 and CH2O protons are effectively removed, and the spectrum of PME and PDE is significantly enhanced. RESULTS: The sensitivity of this pulse sequence has been demonstrated with milk and mouse brain samples. A soft-HSQC spectrum, where only PME and PDE signals appear, can be recorded from these biological samples in minutes with remarkably high signal-to-noise ratio. CONCLUSION: This pulse sequence provides a new and quick method for in vivo studies of phosphorus metabolite in the human brain and other tissues for medical purposes.

Improve the selectively refocused INEPT pulse sequence for detecting phosphomonoesters and phosphodiesters.

In application of the (31)P selectively refocused insensitive nuclei enhanced polarization transfer (srINEPT) technique to the detection of phosphomono- and diesters in tissues, homonuclear couplings between the CH(2)O protons and the NCH(2) protons seriously attenuate the sensitivity. These couplings can be conventionally removed by two soft 180° pulses in the (1)H evolution period which selectively invert the NCH(2) magnetizations. However, the srINEPT pulse sequence can be simplified by replacing the pulse train "soft 180°-hard 180°-soft 180°" with a single soft 180° pulse that selectively inverts the CH(2)O magnetizations. Theoretical analysis in this study demonstrates the correctness of this approach in principle. Validation on a milk phantom allowed us to investigate and discuss advantages and disadvantages of the proposed srINEPT with respect to the original srINEPT. Furthermore, comparison of different selective pulses made it possible to demonstrate that the proposed srINEPT experiment is not sensitive to errors in pulse length, offset, and B(1) field strength of the selective pulse when ReBurp pulse is used for selective refocusing.

Fast detection of choline-containing metabolites in liver using 2D ¹H-¹4N three-bond correlation (HN3BC) spectroscopy.

Detection and quantification of total choline-containing metabolites (CCMs) in tissues by magnetic resonance spectroscopy (MRS) has received considerable attention as a biomarker of cancer. Tissue CCMs are mainly choline (Cho), phosphocholine (PCho), and glycerophosphocholine (GPCho). Because the methyl (1)H resonances of tissue CCMs exhibit small chemical shift differences and overlap significantly in 1D (1)H MRS, quantification of individual components is precluded. Development of a MRS method capably of resolving individual components of tissue CCMs would be a significant advance. Herein, a modification of the 2D (1)H-(14)N HSQC technique is targeted on the two methylene (1)H in the CH(2)O group ((3)J(1H14N)=2.7 Hz) and applied to ex vivo mouse and human liver samples at physiological temperature (37°C). Specifically, the (1)H-(14)N HSQC technique is modified into a 2D (1)H-(14)N three-bond correlation (HN3BC) experiment, which selectively detects the (1)H of CH(2)O coupled to (14)N in CCMs. Separate signals from Cho, PCho, and GPCho components are resolved with high detection sensitivity. A 2D HN3BC spectrum can be recorded from mouse liver in only 1.5 min and from human carcinoma liver tissue in less than 3 min with effective sample volume of 0.2 ml at 14.1T.

A selective NMR method for detecting choline containing compounds in liver tissue: the 1H-14N HSQC experiment.

The feasibility of a (1)H-(14)N HSQC experiment on tissues is demonstrated with a mouse liver based on the J couplings between the protons and the quadrupolar nucleus (14)N in choline. Free choline, phosphocholine, and glycerolphosphocholine (1)H-(14)N HSQC signals were selectively observed with all unwanted signals cleanly suppressed. The CH2O signals were well resolved in the two-dimensional spectrum, which can be used for quantitative analyses.

1H-14N HSQC detection of choline-containing compounds in solutions.

Choline nitrogen ((14)N) has a long relaxation time (seconds) which is due to the highly symmetric chemical environments. (14)N in choline also has coupling constants with protons (0.6 Hz to methyl protons, 2.7 Hz to CH(2)O protons and 0.2 Hz to NCH(2) protons). Based on these properties, we introduce a two-dimensional NMR method to detect choline and its derivatives in solutions. This method is the (1)H-(14)N hetero-nuclear single-quantum correlation (HSQC) experiment which has been developed in solid-state NMR in recent years. Experiments have demonstrated that the (1)H-(14)N HSQC technique is a sensitive method for detection of choline-containing compounds in solutions. From 1mM choline solution in 16 min on a 500 MHz NMR spectrometer, a (1)H-(14)N HSQC spectrum has been recorded with a signal-to-noise ratio of 1700. Free choline, phosphocholine and glycerophosphocholine in milk can be well separated in (1)H-(14)N HSQC spectra. This technique would become a promising analytical approach to mixture analyses where choline-containing compounds are of interest, such as tissue extracts, body fluids and food solutions.

Gridding and fast Fourier transformation on non-uniformly sparse sampled multidimensional NMR data.

For multidimensional NMR method, indirect dimensional non-uniform sparse sampling can dramatically shorten acquisition time of the experiments. However, the non-uniformly sampled NMR data cannot be processed directly using fast Fourier transform (FFT). We show that the non-uniformly sampled NMR data can be reconstructed to Cartesian grid with the gridding method that has been wide applied in MRI, and sequentially be processed using FFT. The proposed gridding-FFT (GFFT) method increases the processing speed sharply compared with the previously proposed non-uniform Fourier Transform, and may speed up application of the non-uniform sparse sampling approaches.

Optimized quantitative DEPT and quantitative POMMIE experiments for 13C NMR.

In quantitative analysis, inverse gated (1)H decoupled (13)C NMR provides higher resolution than (1)H NMR. However, due to the lower sensitivity and longer relaxation time, (13)C NMR experiment takes much longer time to obtain a spectrum with adequate signal-to-noise ratio. The sensitivity can be enhanced with DEPT and INEPT approaches by transferring polarization from (1)H (I) to (13)C (S), but since the enhancements depend on coupling constants ( (1) J SI) and spin systems (SI, SI 2, SI 3), the enhancements for different spin systems are not uniform and quantitative analyses are seriously affected. To overcome these problems, Henderson proposed a quantitative DEPT (Q-DEPT) method by cycling selected read pulse angles and polarization-transfer delays (Henderson, T. J. J. Am. Chem. Soc. 2004, 126, 3682-3683), and satisfactory results for SI system are achieved. However, the optimization is incomplete for the SI 2 and SI 3 systems. Here, we present an improved version of Q-DEPT (Q-DEPT (+)) and a quantitative POMMIE (Q-POMMIE) where the cyclic delays and read pulse phases are applied. The improved methods prove to be suitable for all spin systems over a large J-coupling range (90-230 Hz), and the (13)C signals are nearly equally enhanced with standard deviation less than 5%.

Fast nuclear magnetic resonance correlation spectroscopy without diagonal peaks: the "2-1" correlation spectroscopy.

A fast NMR experiment is proposed for measuring correlation spectroscopy (COSY) spectra with diagonal peaks completely removed or largely suppressed. This new pulse sequence is based on double quantum spectroscopy but with delayed detection, so that the double quantum coherence is effectively transferred to single quantum coherence. Therefore, the pulse sequence can be named "2-1" COSY.

Multiple quantum correlated spectroscopy revamped by asymmetric z-gradient echo detection signal intensity as a function of the read pulse flip angle as verified by heteronuclear 1H/31P experiments.

Heteronuclear multiple quantum (n=+/-0 and n=+/-2) correlated spectroscopy revamped by asymmetric z-gradient echo detection (CRAZED) experiments were performed on the spins 31P and 1H in a H3PO4 solution in order to determine the optimum flip angle for the read pulse. It has been shown that for the negative quantum signals, the maximum signals appear at beta=0, and for the positive quantum signals, the maximum signals appear at beta=pi. The CRAZED signals were compared to the single quantum signals in two-pulse two-gradient experiments. It is found that the CRAZED signals can also be distinguished into gradient echoes and spin echoes. The gradient-echo-type CRAZED signal requires beta=0 and the spin-echo-type CRAZED signal requires beta=pi for maximum echo intensities, in the same way as in single quantum experiments.

NMR study of the folding-unfolding mechanism for the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG).

Hydrogen exchange rates of the imino protons of the thrombin-binding 15 mer DNA aptamer d(G(1)G(2)T(3)T(4)G(5)G(6)T(7)G(8)T(9)G(10)G(11)T(12)T(13)G(14)G(15)) in the presence of Sr(2+) were measured. In the temperature range 15-35 degrees C, the exchange rates of the eight iminos in the quadruplex core were not uniform, with the G(2), G(11) and G(15) iminos exchanging faster, the G(1), G(5), G(10) and G(14) iminos exchanging slower, and the G(6) imino exchanging at a medium rate. In the quadruplex G(1), G(5), G(10) and G(14) adopted syn glycosidic conformation, while G(2), G(6), G(11) and G(15) adopted anti-conformation. It was found that the four slowly exchanging iminos, which were all the syn-iminos, happened to be located in the TT loops that were not easy to open to the solvent. The anti-iminos exchanged faster, but the G(6) imino exchanged slower than other anti-iminos, because its hydrogen bond with the G(10)O6 was stabilized by the TGT loop. The fact that the G(6) imino exchanged at a faster rate than those syn-iminos in the TT loops suggested that the TGT loop was less stable than the TT loops. Unfolding mechanism for the quadruplex was thus proposed: The quadruplex first uncoupled the three base pairs: G(1)-G(15), G(2)-G(14) and G(5)-G(11), which were not protected by any loops. Then it opened the TGT loop. Finally, it opened the TT loops and the sequence became an unstructured random coil that exchanged with the quadruplex conformation. The conformational exchange between the quadruplex and random coil had been detected.

Line shape analyses for water 17O NMR quintet observed in a bacteriophage Pf1 solution at different temperatures.

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.

NMR study on the low-affinity interaction of human serum albumin with diclofenac sodium.

The low-affinity interaction between human serum albumin (HSA) and Diclofenac sodium (DCF) was studied using NMR techniques. Both 13C-NMR chemical shift and linewidth show that the dichlorophenyl ring in DCF molecule plays a primary role in its interaction with HSA. Langmuir adsorption isotherm was applied to evaluate the association constant K and the number of binding sites n of the drug/HSA complex through (1)H-NMR spin-lattice relaxation measurement. The results indicate that Langmuir isotherm can perfectly explain the capacity of low-affinity binding of proteins for the ligands.

[The synthesis, 1H NMR and IR study of [(n-Bu)4N]2[Mo2O5(OC10H6O)2] and [(n-Bu)4N]2[Mo4O10 (OC10H6O)2(OCH3)2]].

The title organo-molybdate derivatives are synthesized and their IR, 1H NMR spectra have been determined and the relations between the structures and the 1H NMR and IR parameters have been studied. The results indicate that the red shift of the IR frequency of Mo-O-Mo in [(n-Bu)4N]2[Mo2O5(OC10H6O)2] (complex I) takes place to compare with that in [(n-Bu)4N]2[Mo4O10 (OC10H6O)2(OCH3)2] (complex II) and lower filed shift of 1H NMR of the aromatic H atoms in complex II occurs as contrasted to that in the complex I. It is found also the organo-molybdate derivatives are very sensitive to the acidity of the chemical system.

Conformational Flexibility of Deoxyoligonucleotide d(GGTATACC)(2) in Water.

AT-rich deoxyoligonucleotide provides a binding site possibly at the minor groove for some anti-tumor drugs by hydrophobic or Van der Waals interactions. In this paper, it is demonstrated by study of d (GGTATACC)(2) that the DNA-drug interaction may be dependent on the structural flexibility at the minor groove. The solution structure of d (GGTATACC)(2) in water is described by distance-restrained molecular dynamics calculation and it is suggested that d (GGTATACC)(2) in solution maintains the double helix of B-type with trans conformations of base to sugar and C2'-endo conformation for the deoxyribose ring. It is found that the end moieties GGT and ACC are relatively rigid while T(5) residue is flexible, which may account for the activity of the minor groove.

Study of hydrolytic kinetics of the hexachlorostannate anion by two-dimensional 119 Sn exchange spectroscopy.

Two-dimensional 119 Sn exchange spectroscopy was applied to study the hydrolytic kinetics of the hexachloro-stannate anion, SnCl6 2- . Two 119 Sn exchange spectra for SnCl4 · 5H2 O solutions with various HCl concentrations are presented. Magnetization exchanges, which are related to the hydrolysis processes, occur in the SnCl6 2- hydrolysis series. The exchange rate constants, the hydrolysis rate constants and the hydrolysis equilibrium constants were evaluated.