Heteronuclear Editing Research Articles

Inverse detection and heteronuclear editing in 1H-15N correlation and 1H-1H double-quantum NMR spectroscopy in the solid state under fast MAS.

Signal enhancement in heteronuclear correlation spectra as well as signal selection in 1H experiments can be achieved through inverse, i.e., 1H, detection in the solid state under fast MAS conditions. Using recoupled polarization transfer (REPT), a heteronuclear 1H-15N single-quantum correlation (HSQC) experiment is presented whose symmetrical design allows the frequency dimensions to be easily interchanged. By observing the 15N dimension indirectly and detecting on 1H, the sensitivity is experimentally found to be increased by factors between 5 and 10 relative to conventional 15N detection. In addition, the inverse 1H-15N REPT-HSQC scheme can be readily used as a filter for the 1H signal. As an example, we present the combination of such a heteronuclear filter with a subsequent 1H-1H DQ experiment, yielding two-dimensional 15N-edited 1H-1H DQ MAS spectra. In this way, specific selection or suppression of 1H resonances is possible in solid-state MAS experiments, by use of which the resolution can be improved and information can be unravelled in 1H spectra.

Heteronuclear editing method for detecting NOE between chemically equivalent protons: application to polycyclic aromatic hydrocarbons

The complete characterization of polycyclic aromatic hydrocarbons (PAHs) and tetrasubstituted ethylenes is critical to an understanding of their reactivity, for which NMR is an important tool. Chemical shifts can provide a direct measure of charge distribution and aromaticity. Unfortunately, COSY, NOESY and heteronuclear correlation cannot provide a complete assignment of the NMR spectra for some carbon-rich PAHs with symmetrical bay regions. The protons in the bay regions would yield NOE signals if they were not symmetrical. Natural substitution of 12C with 13C can be used to break the symmetry and yield these useful NOE signals. Using gradient-assisted editing, unequivocal assignments have been achieved for some previously problematic molecules. Copyright © 2000 John Wiley & Sons, Ltd.

Heteronuclear editing method for detecting NOE between chemically equivalent protons: application to polycyclic aromatic hydrocarbons

Heteronuclear editing method for detecting NOE between chemically equivalent protons: application to polycyclic aromatic hydrocarbons

Spectroscopic imaging of glutamate C4 turnover in human brain.

One-dimensional spectroscopic imaging of (13)C-4-glutamate turnover is performed in the human brain with a 6 cc nominal voxel resolution at 4T. Data were acquired with an indirect detection approach using a short spin echo single quantum (1)H-(13)C heteronuclear editing method and a 7 cm surface coil with quadrature (13)C decoupling coils. To analyze the data as a function of tissue type, T(1)-based image segmentation through the surface coil was performed to determine the gray and white matter contributions to each voxel. The tricarboxylic acid (TCA) cycle rate in gray and white matter was then determined using a two-compartment model with the tissue fractionation derived from the image segmentation. The mean values for the TCA cycle rate for occipital gray and white matter from three volunteers was 0.88 +/- 0.12 and 0.28 +/- 0.13 respectively, in agreement with literature data.

In Vivo Detection of 15N-Coupled Protons in Rat Brain by ISIS Localization and Multiple-Quantum Editing

Three-dimensional image-selected in vivo spectroscopy (ISIS) was combined with phase-cycled 1H–15N heteronuclear multiple-quantum coherence (HMQC) transfer NMR for localized selective observation of protons J-coupled to 15N in phantoms and in vivo. The ISIS–HMQC sequence, supplemented by jump–return water suppression, permitted localized selective observation of 2–5 μmol of [15Nindole]tryptophan, a precursor of the neurotransmitter serotonin, through the 15N-coupled proton in 20–40 min of acquisition in vitro at 4.7 T. In vivo, the amide proton of [5-15N]glutamine was selectively observed in the brain of spontaneously breathing 15NH4+-infused rats, using a volume probe with homogeneous 1H and 15N fields. Signal recovery after three-dimensional localization was 72–82% in phantoms and 59 ± 4% in vivo. The result demonstrates that localized selective observation of 15N-coupled protons, with complete cancellation of all other protons except water, can be achieved in spontaneously breathing animals by the ISIS–HMQC sequence. This sequence performs both volume selection and heteronuclear editing through an addition/subtraction scheme and predicts the highest intrinsic sensitivity for detection of 15N-coupled protons in the selected volume. The advantages and limitations of this method for in vivo application are compared to those of other localized editing techniques currently in use for non-exchanging protons.

Unambiguous NOE assignments in proteins by a combination of through-bond and through-space correlations

Heteronuclear editing has found widespread use in the detection ofproton–proton dipolar interactions in isotopically labelled proteins.However, in cases where both the resonances of protons and directly bound13C or 15N spins of two or more sites aredegenerate, unambiguous assignments are difficult to obtain by conventionalmethods. Here, we present simple extensions of well-known triple-resonancepulse sequences which improve the dispersion of NOESY spectra. In order torecord the chemical shifts of backbone nuclei which allow a resolution ofoverlapping cross peaks, the magnetization is relayed via the scalarcoupling network either before or after the NOE mixing period. The novelpulse sequences are applied to flavodoxin from the sulfate-reducing organismDesulfovibrio vulgaris. A number of previously unassigned NOE interactionsinvolving α-, β- and amide protons can be unequivocallyidentified, suggesting that the accuracy of protein structure determinationcan be improved.

Magnetization exchange network editing: mathematical principles and experimental demonstration

Abstract We have examined analytically the effects of different NMR magnetization exchange network editing (MENE) procedures, starting from equilibrium or nonequilibrium conditions, on generalized systems that undergo magnetization exchange (cross-relaxation and/or chemical exchange). Larger errors in derived exchange rate constants are found in experiments that start from nonequilibrium states than in those that start from equilibrium. On the other hand, editing experiments that decompose the dynamic matrix into two or more block diagonal submatrices generally yield more accurate exchange rate constants. Theoretical arguments, backed up by experimental results, demonstrate that MENE experiments lead to increased rates of relaxation for the edited cross and diagonal peaks. We describe a new class of heteronuclear editing experiment in which the system starts from a nonequilibrium state and is edited during the mixing period. In an experimental application of this approach to the spectral analysis of a small protein, recombinant human ubiquitin (M r 8565) labeled uniformly with 15 N and 13 C, we demonstrate how the cross-relaxation network of the protein can be decomposed simultaneously into subnetworks of 15 N-bound protons, aliphatic 13 C-bound protons, aromatic 13 C-bound protons, and ( 12 C/ 14 N/O)-bound protons. Such a decomposition permits the measurement of slower magnetization exchange rates, including those that are masked in conventional cross-relaxation experiments.

Solution Structure of Bound Trimethoprim in Its Complex with Lactobacillus casei Dihydrofolate Reductase

Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 1:1 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.

Motion-Insensitive Localized 13C Spectroscopy Using Cyclic and Slice-Selective J Cross Polarization

Several new methods are proposed for the sensitive localized detection of 13C nuclei on the basis of cyclic and slice-selective J cross polarization in 13C 1H x spin systems. The 13C nuclei are detected either directly after the amplitude is enhanced by polarization transfer in the rotating frame or, preferably, indirectly by heteronuclear editing of signals of the 1H nuclei coupled to 13C. In the latter case, the sensitivity corresponds to that of 1H rather than to that of 13C resonance. Test experiments are reported. In vitro applications to a hen egg and a fresh porcine shank prove the applicability of the methods to biological objects with 13C in natural abundance. A particular advantage of the new rotating-frame methods over laboratory-frame techniques serving the same purpose is the insensitivity to motions of the object. This is demonstrated by experiments with a moving sample. Hartmann/Hahn mismatch can be compensated using the MOIST modification. The time-averaged absorbed radiofrequency power per kilogram body weight was estimated on the basis of a model for surface power absorption. The result lies well below the standard safety limits for clinical applications.

Proton-detected 13C imaging using cyclic J cross polarization

A new method is proposed permitting two- or three-dimensional Fourier transform imaging of protons coupled to 13C nuclei. In this way the spatial distribution of a preselected 13C resonance line can be imaged indirectly. The initial excitation as well as the detection of the signal are performed in the proton radiofrequency channel so that the utmost sensitivity is achieved. The principle is cyclic J cross polarization (CYCLCROP) for heteronuclear editing combined with a standard imaging sequence. The sequence has been implemented on a 4.7 T tomography system and successfully tested with a phantom as well as with biological objects with 13C in natural abundance.

Adiabatic J Cross Polarization for Localized Direct and Proton-Detected 13C Spectroscopy

This work predominantly refers to in vivo spectroscopy. The objective is to record volume-selectively 13C signals by the aid of a new, simple, and robust localization technique. J cross polarization (or coherent rotating-frame polarization transfer) between coupled 1H and 13C nuclei is employed in the adiabatic level-crossing version. In the presence of a magnetic field gradient, the passage of the Hartmann/Hahn-matched energy splitting depends on position so that the polarization-transfer process is slice-selective. The signal enhancement of rare X nuclei thus is performed slice-selectively. Test experiments demonstrating the efficiency of adiabatic J cross-polarization-localized spectroscopy (AJCP-LOSY) are reported. The cyclic application of the AJCP-LOSY procedure permits the indirect slice-selective detection of coupled X nuclei (adiabatic CYCLCROP-LOSY). Signals from coupled nuclei are thus observed while other signals are suppressed. The technique can be extended to volume-selective excitations of 13C or, preferably, of 1H nuclei coupled to 13C. The latter proposal implies heteronuclear editing analogous to the CYCLPOT-VOSING methods for laboratory-frame polarization transfer previously suggested.

J Cross-Polarization Methods for Localized NMR without Magnetic Field Gradients

This work predominantly refers to 13C in vivo spectroscopy and has two objectives. First, heteronuclear editing is considered for proton-detected NMR spectroscopy of rare nuclei. The second aspect refers to localized heteronuclear NMR spectroscopy implying the editing option. Both purposes are achieved by employing J cross polarization (or coherent rotating-frame polarization transfer). The spatial selectivity is due to different in-homogeneities of the superimposed double-resonance radiofrequency fields of the J cross-polarization experiment. A 13C surface coil, for instance, was combined with a 1H birdcage resonator, so that J cross polarization is restricted to the region where the Hartmann/Hahn condition is approached. Heteronuclear editing of NMR lines for proton-detected 13C spectroscopy was performed by the aid of a cyclic cross-polarization pathway. Test experiments demonstrating the volume selectivity of the transfer process and the editing efficiency of the cyclic cross-polarization procedure are reported.

Cyclic and Slice-Selective J Cross Polarization for Heteronuclear Editing and Localized NMR

This work has two objectives. First, heteronuclear editing was considered, with particular aim at proton-detected NMR spectroscopy of rare nuclei. The second aspect refers to localized heteronuclear NMR spectroscopy, implying the editing option. The common basis of both techniques is J cross polarization (or coherent rotating-frame polarization transfer). In the presence of magnetic field gradients this process is slice selective and is introduced as a principle for localized liquid-state NMR, particularly of heteronuclear spins. Heteronuclear editing of NMR lines is performed by the aid of a cyclic cross-polarization pathway. Both methods can be combined to a technique for localized proton-detected NMR spectroscopy of rare nuclei. Test experiments demonstrating the slice selectivity of the transfer process and the efficiency of the cyclic cross-polarization editing procedure are reported. Applications to localized homo- or heteronuclear spectroscopy and imaging are discussed.

Heteronuclear spectral editing with adiabatic pulses

Spectral editing pulse sequences have greatly expanded the capabilities of NMR spectroscopy in many different areas of research. In the field of in vivo NMR, editing is frequently employed to extract spectral information which may otherwise be concealed by broad and overlapping resonances. Heteronuclear ( i3C‘H) spectral editing (1-6) provides the additional capability to monitor the metabolism of i3C-labeled compounds while exploiting the high sensitivity of ‘H observation. Recent studies have shown that heteronuclear editing of 13C signals has many advantages, including a more than lo-fold increase in signal-to-noise, over direct 13C observation with nuclear Overhauser enhancement ( 7). Existing spectral editing pulse sequences are based upon conventional radiofrequency pulses which induce flip angles that are directly proportional to the RF field strength, B, . This hip-angle dependence on Br can result in substantial signal losses when an inhomogeneous RF coil, such as a surface coil, is used for pulse transmission. Even with homogeneous RF coils, minor misadjustment of flip angles can cause significant signal losses in multiphase editing sequences since the net error accumulates in a multiplicative manner with each pulse. In this Communication, we describe a pulse which achieves uniform heteronuclear spectral editing above a threshold Br where the pulse operates adiabatically. This Brinsensitive spectral editing pulse (BISEP) selectively excites ‘H spins engaged in heteronuclear J modulation. Uncoupled spins are initially rotated into the transverse plane, but then are returned to the longitudinal axis by the end of the pulse. Theoretically, spectral editing is complete in a single acquisition. BISEP is based on our adiabatic pulse, BIR-4, which can induce any desired flip angle with compensation for Br inhomogeneity and resonance offset (8, 9). The flip angle generated by BIR-4 is determined by two discontinuous phase shifts, Ab, and A&, which take place at specific times during the pulse when the magnetization has been adiabatically rotated onto the transverse plane. For an arbitrary flip angle 8 to be induced at the completion of the pulse, the discontinuous phase shifts are A4, = ?r + 0 / 2 and A& = ( 7r + 6 / 2 ) . These expressions can be equated to give A4 1 A& = 2a + 0 = 8. If A& = A$,, this latter equation predicts that BIR4 will execute a B,-insensitive identity transformation (fl = 0) provided the pulse is operating adi-

Motion‐insensitive volume‐selective pulse sequences for direct and proton‐detected 13C spectroscopy: Detection of glycogen in the human liver in vivo

Two compact pulse sequences are reported for the volume-selective detection of 13C nuclei. 13C signals are either directly acquired after enhancing the amplitude by polarization transfer from the coupled protons or, preferably, indirectly detected by heteronuclear editing of proton signals of 1H nuclei coupled to 13C. In the latter case, the full sensitivity of proton resonance is achieved, and signals from uncoupled protons or protons coupled to non-13C nuclei or 13C nuclei of undesired compounds are suppressed. Both sequences are single-scan procedures and are insensitive to the pulse phases and to motions of the investigated organs. The insensitivity to organ motions is due to the extremely compact character of these sequences avoiding coherence evolution periods as far as possible. This is achieved first by the introduction of double-resonance sandwich (DORSA) pulses accomplishing the polarization transfer slice selectively in a very short time. The second reason is that the initial evolution interval which is usually part of polarization transfer experiments is avoided by the aid of a doublet line-selective inversion pulse. Several test experiments in vivo are reported. In particular, it is demonstrated that glycogen can be detected in natural abundance in the human liver in vivo using a 2-T whole-body tomograph even without the use of heart or respiration triggers. The total acquisition time was 22 min, and a signal-to-noise ratio of 6 was achieved.

Heteronuclear filters in two-dimensional [1H, 1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions

The use of heteronuclear filters enables the editing of complex 1H nuclear magnetic resonance (NMR) spectra into simplified subspectra containing a lesser number of resonance lines, which are then more easily amenable to detailed spectral analysis. This editing is based on the creation of heteronuclear two-spin or multiple-spin coherence and discrimination between protons that do or do not participate in these heteronuclear coherences. In principle, heteronuclear editing can be used in conjunction with one-dimensional or multidimensional 1H-NMR experiments for studies of a wide variety of low-molecular-weight compounds or macromolecular systems, and is implicitely applied in a wide range of heteronuclear NMR experiments with proton detection (e.g. Bax et al. 1983; Griffey &amp; Redfield, 1987). In the present article we shall focus on the use of heteronuclear filters in two-dimensional (2D) [1H, 1H]-NMR experiments. The selection of the material covered was primarily motivated by its impact on the practice of protein structure determination in solution, and on NMR studies of intermolecular interactions with biological macromolecules. Section 2 surveys potential applications of heteronuclear filters in this area. The remainder of the article is devoted to an introduction of the theoretical principles used in heteronuclear filters, and to a detailed description of the experimental implementation of these measurements. In writing the review we tried to minimize redundancy with the recent article in Quarterly Review of Biophysics by Griffey &amp; Redfield (1987) and to concentrate on experiments that were introduced during the period 1986–9.

Indirect 13C tomography and volume-selective spectroscopy via proton NMR. II. Imaging

Pulse sequences are presented permitting two- or three-dimensional Fourier transform imaging selectively of protons coupled to 13C nuclei. In this way, 13C images can indirectly be recorded with proton sensitivity. The maximum enhancement theoretically is a factor of 64 times the number of protons coupled to a 13C nucleus. The techniques are based on the heteronuclear editing sequences for volume-selective spectroscopy, which are reported in a previous article. The heteronuclear multiple-quantum filter imaging variant has been implemented on 4.7 and 2 T NMR tomographs and tested with phantoms as well as with biological objects with 13C in natural abundance.

DENSITY OPERATOR FORMALISM FOR SPIN SYSTEMS WITH PERMUTATION SYMMETRY IN MULTIPLE PULSE AND 2D NMR EXPERIMENTS (Ⅱ)——MULTIPLE QUANTUM PRODUCT OPERATOR FORMALISM

Based on the Symmetry-Adapted Product Operator (SPO), the Moltiple Quantum Coherence Product Operator (MQCPO) formalism is introduced in this paper. The modified density operator theory is universal for the description of modern multiple pulse and two-dimensional NMR experiments performed on InS(I = 1/2, 5 = 1/2; n = an arbitray integer number) spin systems. In fact, MQCPO is a Hermitian combination of irredusible tensor operators for a compsite particle, and reveals symmetry properties of the spin system in a different aspect from SAPO. There exist simple linear relations between MQCPO and SAPO for In(I = 1/2, n = 2,3) spin systems, the SAPO forms of MQCPO are listed as a usual example. MQCPO shows a simple cyclic commutation relationship with the free procession Hamiltonian, but not with the Hamiltonian of a pulse effect. Therefore, MQCPO is convenient for description of evolution period. Using the linear relations between MQCPO and SAPO, as well as the permutation properties for the coordinate indices of Cartesian components in a SAPO, the description of a pulse action tarns out to be easy and direct under the "z"-representation. Analyses of the pulse sequences for heteronuclear spectra editing and spin topology filtration have shown the power of this formalism.

Extended heteronuclear editing of 2D 1H NMR spectra of isotope-labeled proteins, using the X( ω1, ω2) double half filter

Extended heteronuclear editing of 2D 1H NMR spectra of isotope-labeled proteins, using the X( ω1, ω2) double half filter

Isotope-detected 1H NMR studies of proteins: a general strategy for editing interproton nuclear Overhauser effects by heteronuclear decoupling, with application to phage lambda repressor.

A strategy for editing interproton nuclear Overhauser effects (NOEs) in proteins is proposed and illustrated. Selective incorporation of 13C- (or 15N)-labeled amino acids into a protein permits NOEs involving the labeled residues to be identified by heteronuclear difference decoupling. Such heteronuclear editing simplifies the NOE difference spectrum and avoids ambiguities due to spin diffusion. Isotope-detected 1H NMR thus opens to study proteins too large for conventional one- and two-dimensional NMR methods (20-75 kDa). We have applied this strategy to the N-terminal domain of phage lambda repressor, a protein of dimer molecular mass 23 kDa. A tertiary NOE from an internal aromatic ring (Phe-51) to a beta-13C-labeled alanine residue (Ala-62) is demonstrated.