Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

Application of biselective refocusing soft pulses to the simplification of heteronuclear correlation spectra

The biselective spin echo technique allows the signals of coupled proton pairs to be extracted from crowded liquid state proton NMR spectra. Its use as a preparation sequence in heteronuclear chemical shift correlation experiments requires the removal of the heteronuclear coupling interaction during the biselective echo time. The discrimination between coupled and uncoupled protons signals is achieved by double quantum filtration, which delivers antiphase magnetization states. The latter are not directly compatible with the design of an HSQC-like pulse sequence. The conversion of antiphase to in-phase magnetization states by a second biselective echo sequence solves this problem. The optimization of spin echo delays is also discussed. Lastly, the article presents modified HSQC and HMBC pulses sequences in which information is obtained solely for the biselectively selected proton pairs. A peracetylated trisaccharide was used as a test molecule.     

Composite Pulsed Field Gradients with Refocused Chemical Shifts and Short Recovery Time

An improved self-compensating pulsed field gradient (PFG) technique that combines antiphase gradient pairs with broadband frequency-modulated 180° pulses is proposed. The antiphase gradient pairs lead to superb system recovery. In addition, evolution under chemical shift and heteronuclearJcoupling are refocused during the PFG, making it appear effectively instantaneous. This new approach makes it possible to obtain high-resolutionphase-sensitive2D spectra for the PFG version of many experiments such as COSY, DQF-COSY, and HSQC without adding extra compensating delays or pulses. While reasonable suppression of unwanted magnetization is achieved, this method also gives satisfactory retention of desired signals. As a bonus, the field-frequency lock is not perturbed during the experiments.     

一个获得压水峰良好效果的简单方法

蛋白质NMR中最基本的实验是HSQC实验．在HSQC实验中,水峰压制的主要方法之一是利用一对与异核旋磁比比值相适应的梯度脉冲．该文介绍一种简单易行的方法,只需对这一对脉冲进行适当的延长便能使得水峰几乎完全消失.     

Compensation of refocusing inefficiency with synchronized inversion sweep (CRISIS) in multiplicity-edited HSQC

Use of adiabatic pulses in broadband inversion and decoupling is well known. Replacement of the rectangular pi pulses in the INEPT and rev-INEPT parts of the HSQC and gHSQC experiments with adiabatic pulses substantially improves the sensitivity of these experiments. However, modulation of cross peak intensity in multiplicity-edited HSQC or gHSQC experiments can be quite severe. These modulations arise during the multiplicity-editing periods due to the inefficient refocusing of the spin-echo caused by the mismatch of the echo delay with the one-bond coupling constant. These modulations (which we call echo modulations) are field strength (and hence spectral width) independent. Use of adiabatic pulses with the inversion sweep synchronized to the 1H-13C coupling constant range typically observed in a 13C spectrum will provide substantial improvement in sensitivity. The inversion profile problems associated with rectangular pi pulses can be moderately compensated by composite pulse schemes and these schemes could prove to be reasonable alternatives to adiabatic pulses. However, the adiabatic sweep provides a unique method to compensate the echo modulations for multiplicity-edited experiments. The origin and the compensation of refocusing inefficiency with synchronized inversion sweep (CRISIS) method to minimize these modulations is described.     

Sensitivity-enhanced IPAP experiments for measuring one-bond C–C and C–H residual dipolar couplings in proteins

Sensitivity-enhanced 2D IPAP experiments using the accordion principle for measuring one-bond 13C'-13Calpha and 1Halpha-13Calpha dipolar couplings in proteins are presented. The resolution of the resulting spectra is identical to that of the decoupled HSQC spectra and the sensitivity of the corresponding 1D acquisitions are only slightly lower than those obtained with 3D HNCO and 3D HN(COCA)HA pulse sequences due to an additional delay 2Delta. For cases of limited resolution in the 2D 15N-1HN HSQC spectrum the current pulse sequences can easily be modified into 3D versions by introducing a poorly digitized third dimension, if so desired. The experiments described here are a valuable addition to the suites available for determination of residual dipolar couplings in biological systems.     

Sensitivity-enhanced IPAP experiments for measuring one-bond 13C'-13Calpha and 13Calpha-1Halpha residual dipolar couplings in proteins

Sensitivity-enhanced 2D IPAP experiments using the accordion principle for measuring one-bond 13C'-13Calpha and 1Halpha-13Calpha dipolar couplings in proteins are presented. The resolution of the resulting spectra is identical to that of the decoupled HSQC spectra and the sensitivity of the corresponding 1D acquisitions are only slightly lower than those obtained with 3D HNCO and 3D HN(COCA)HA pulse sequences due to an additional delay 2Delta. For cases of limited resolution in the 2D 15N-1HN HSQC spectrum the current pulse sequences can easily be modified into 3D versions by introducing a poorly digitized third dimension, if so desired. The experiments described here are a valuable addition to the suites available for determination of residual dipolar couplings in biological systems.     

Comparison of the Effects of Different <Superscript>19</Superscript>F <Emphasis Type="Italic">π</Emphasis> Pulses on the Sensitivity and Phaseability of the <Superscript>19</Superscript>F-<Superscript>13</Superscript>C HSQC

The ¹⁹F-¹³C heteronuclear single quantum coherence (HSQC) experiment is vital for the structural elucidation of polyfluorinated organic species, yet its sensitivity and phaseability are limited by difficulties in uniform excitation of the widely disperse ¹⁹F spectral window. Adiabatic pulses of different types have previously been employed to generate effective π pulses for inversion and refocussing, but a systematic comparison of various adiabatic and other inversion pulses has not been published. In this work, it was observed that the use of a broadband inversion pulse (BIP) during the t ₁ evolution period resulted in properly phaseable spectra for experiments optimized to detect ¹ J _CF, in contrast to CHIRP or WURST adiabatic pulses. For the INEPT and reverse-INEPT transfer segments of the HSQC, optimal sensitivity for resonances distant from the transmitter frequency was afforded by optimized universal rotation (BURBOP) or CHIRP pulses. In HSQC experiments with delays optimized for two-bond correlations, only the use of BURBOP pulses in INEPT and reverse-INEPT sequences afforded spectra cleanly phaseable across the F ₂ and F ₁ spectral windows. This observation is supported by off-resonance pulsed field gradient spin-echo experiments.     

自旋1/2-自旋1耦合系统的HSQC和HDQC实验的研究

对自旋1/2-自旋1耦合系统的单量子HSQC和双量子HDQC脉冲序列进行了理论和实验的探讨. 研究结果表明,文献提出的HDQC实验中实际上还发生了零量子跃迁,因此该实验应该是多量子HMQC实验. 研究结果还表明,HSQC有比HDQC更高的灵敏度.     

Convection-compensating diffusion experiments with phase-sensitive double-quantum filtering

We present a design scheme for phase-sensitive, convection-compensating diffusion experiments with gradient-selected homonuclear double-quantum filtering. The scheme consists of three blocks: a 1/2J evolution period during which antiphase single-quantum coherences are created; a period of double-quantum evolution; and another 1/2J period, during which antiphase single-quantum coherences are converted back into an in-phase state. A single coherence transfer pathway is selected using an asymmetric set of gradient pulses, and both diffusion sensitization and convection compensation are built into the gradient coherence transfer pathway selection. Double-quantum filtering can be used either for solvent suppression or spectral editing, and we demonstrate examples of both applications. The new experiment performs well in the absence of a field-frequency lock and does not require magnitude Fourier transformation. The proposed scheme may offer advantages in diffusion measurements of spectrally crowded systems, particularly small molecules solubilized in colloidal solutions or bound to macromolecules.     

Accurate determination of small one-bond heteronuclear residual dipolar couplings by F1 coupled HSQC modified with a G-BIRD(r) module

We report a G-BIRD(r) modified coupled HSQC experiment for the accurate determination of one-bond heteronuclear residual dipolar couplings. The G-BIRD(r) module has been employed to refocus the long-range coupling evolution of the heteronucleus during the t1 frequency labeling period. As a result, the crosspeaks obtained are split by only the direct one-bond coupling that can be extracted by measuring simple frequency differences between singlet maxima. Additionally the decoupling of long-range multiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence resulting in a significant sensitivity and resolution improvement.     

Sensitivity enhanced recoupling experiments in solid-state NMR by gamma preparation

In this paper, we introduce a class of dipolar recoupling experiments under magic angle spinning (MAS), which use gamma dependent antiphase polarization during the t1 evolution period. We show that this helps us to design dipolar recoupling experiments that transfer both components of the transverse magnetization of spin S to a coupled spin I in the mixing step of a 2D NMR experiment. We show that it is possible to design such transfer schemes and make them insensitive to the orientation dependency of the couplings in powders. This helps us to develop sensitivity enhanced 2D NMR experiments of powder samples under MAS.     

Novel methods for characterizing a decoupler channel using "undetectable" quantum coherences.

Exact solutions for the effect of time-independent RF pulses on any initial configuration of an IS J-coupled system demonstrate that on-resonance CW decoupling yields signals whose frequency depends on RF field strength and homogeneity. These signals are enhanced starting with "undetectable" antiphase and multiple quantum coherences, which can also produce centerband intensity to mimic the signal from decoupled Sx. Conversely, these coherences can be generated from Sx using a low-power pulse, B1 = J/2, of length (2J)-1, dubbed a "90J pulse" since it is the selective equivalent of {(2J)-1-90[I]}. Utilizing 90J pulses, new characterization-of-decoupler (COD) pulse sequences can determine the performance of an insensitive I-spin channel by observing large signals from either antiphase or multiple quantum coherences with the S-spin channel, allowing, in minutes rather than hours: (i) frequency calibration to an accuracy of 0.1 Hz; (ii) measurement of RF amplitudes over a 500-fold variation; and (iii) mapping of RF homogeneity along the sample axis with a single 1D B1 spectrum. These 90J coherence transfer pulses are of potential general use for selective spectroscopy.     

Measurement of small one-bond proton-carbon residual dipolar coupling constants in partially oriented (13)C natural abundance oligosaccharide samples: analysis of heteronuclear (1)J(CH)-modulated spectra with the BIRD inversion pulse

Two 2D J-modulated HSQC-based experiments were designed for precise determination of small residual dipolar one-bond carbon-proton coupling constants in (13)C natural abundance carbohydrates. Crucial to the precision of a few hundredths of Hz achieved by these methods was the use of long modulation intervals and BIRD pulses, which acted as semiselective inversion pulses. The BIRD pulses eliminated effective evolution of all but (1)J(CH) couplings, resulting in signal modulation that can be described by simple modulation functions. A thorough analysis of such modulation functions for a typical four-spin carbohydrate spin system was performed for both experiments. The results showed that the evolution of the (1)H-(1)H and long-range (1)H-(13)C couplings during the BIRD pulses did not necessitate the introduction of more complicated modulation functions. The effects of pulse imperfections were also inspected. While weakly coupled spin systems can be analyzed by simple fitting of cross peak intensities, in strongly coupled spin systems the evolution of the density matrix needs to be considered in order to analyse data accurately. However, if strong coupling effects are modest the errors in coupling constants determined by the "weak coupling" analysis are of similar magnitudes in oriented and isotropic samples and are partially cancelled during dipolar coupling calculation. Simple criteria have been established as to when the strong coupling treatment needs to be invoked.     

Optimum spin-state selection for all multiplicities in the acquisition dimension of the HSQC experiment

Most conventional heteronuclear spin-state-selective (S(3)) NMR experiments only work for a specific multiplicity, typically IS spin systems. Here, we introduce a general and efficient IPAP strategy to achieve S(3) editing simultaneously for all multiplicities in the acquisition dimension of the HSQC experiment. Complementary in-phase (HSQC-IP) and anti-phase (HSQC-AP) data are separately recorded with a simple phase exchange of two 90 degrees proton pulses involved in the mixing process of the F2-coupled sensitivity-improved HSQC pulse sequence. Additive and subtractive linear combination of these IP/AP data generates simplified F2-alpha/beta-spin-edited HSQC subspectra for all IS, I(2)S, and I(3)S spin systems and combines enhanced and optimized sensitivity with excellent tolerance to unwanted cross-talk contributions over a considerable range of coupling constants. Practical aspects such as pulse phase settings, transfer efficiency dependence, inter-pulse delay optimization, and percentage of cross-talk are theoretically analyzed and discussed as a function of each I(n)S multiplicity. Particular emphasis on the features associated to spin-editing in diastereotopic I(2)S spin systems and application to the measurement of long-range proton-carbon coupling constants are also provided.     

Spatiotemporal antiphase dynamics in coupled extended optical media

Experimental evidence of spatiotemporal antiphase dynamics is given for an extended system made of two liquid crystal slices that are optically coupled by two equal amplitude counterpropagating pumping beams. Theory and experiments carried out in a transverse one-dimensional configuration show that roll patterns are generated in each slice. These rolls are spatially in-phase or antiphase for a focusing or a defocusing nonlinearity type, respectively. These in-phase or antiphase dynamics remain robust even for complex spatiotemporal regimes such as dislocation regimes.     

Methods for the measurement of (1)J(NCalpha) and (2)J(NCalpha) from a simplified 2D (13)C(alpha)-coupled (15)N SE-HSQC spectrum

Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.     

Signal enhancement using 45 degrees water flipback for 3D 15N-edited ROESY and NOESY HMQC and HSQC.

A novel implementation of the water flipback technique employing a 45 degrees flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D 15N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45 degrees water flipback are presented using both HMQC and HSQC for the 15N dimension. The 45 degrees flipback pulse is followed by a gradient, a water selective 180 degrees pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t1 and t2 evolution periods is suppressed using gradients. Water resonance planes from NOESY-HMQC and NOESY-HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein-water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.     

Improved J-compensated sequences based on short composite pulses

Efficient J-compensated sequences that are shorter in duration and use less RF pulses have been created from short but very efficient composite 90 degrees RF pulses. The improved J-compensation transforms in-phase into antiphase magnetization and can be incorporated in any pulse sequence that involves evolution of heteronuclear J-couplings. The compensated sequences were tested and incorporated into an HMBC sequence. J-compensated experiments referred to as HMBC-J45 + 90A and HMBC-J45 + 90B, were found to be effective over a wide range of J values.