More line narrowing in TROSY by decoupling of long-range couplings: shift correlation and 1JNC' coupling constant measurements

Since the introduction of RDCs in high-resolution NMR studies of macromolecules, there is a growing interest in the development of accurate, and sensitive methods for determining coupling constants. Most methods for extracting these couplings are based on the measurement of the splitting between multiplet components in J-coupled spectra. However, these methods are often unreliable since undesired multiple-bond couplings can considerably broaden the multiplet components and consequently make accurate determination of their position difficult. To demonstrate one approach to this problem, G-BIRD((r)) decoupled TROSY sequences are proposed for the measurement of (1)J(NH) and (1)J(NC') coupling constants. Resolved or unresolved splittings due to remote protons are removed by a G-BIRD((r)) module employed during t(1) and as a result, spectra with narrow, well-resolved peaks are obtained from which heteronuclear one-bond couplings can be accurately measured. Moreover, introduction of a spin-state-selective alpha/beta-filter in the TROSY sequence allows the separation of the (1)J(NC') doublet components into two subspectra which contain the same number of peaks as the regular TROSY spectrum. The (1)J(NC') couplings are obtained from the displacement between the corresponding peaks in the subspectra.     

Three-dimensional protein NMR TROSY-type (15)N-resolved (1)H(N)-(1)H(N) NOESY spectra with diagonal peak suppression

An earlier two-dimensional NOESY experiment with diagonal peak suppression in the (1)H(N)-(1)H(N) region is extended to three dimensions by including (15)N evolution while maintaining the TROSY approach throughout. The technique suppresses all anti-TROSY resonances by appropriate pulse sequence elements and for large molecules at high fields possible semi- and anti-TROSY artifacts are further suppressed by virtue of much shorter transverse relaxation times for these components. The new technique is demonstrated using an (15)N-labeled protein sample, RAP 17-97 (N-terminal domain of alpha2-macroglobulin Receptor Associated Protein), in H(2)O at 500 MHz.     

Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(13)C groups in proteins.

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394-6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the (13)C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S(3) TPPI). The new 3D S(3) TPPI TROSY HCCH method is demonstrated on a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.     

J-modulated TROSY experiment extends the limits of homonuclear coupling measurements for larger proteins.

This paper describes the use of a TROSY experimental scheme and its variant extended with a scaled J-modulation spin-echo sequence for accurate and sensitive measurement of homonuclear 3J(H(N)H(alpha)) coupling constants in larger proteins with uniform 15N labeling. Exclusive selection of the most slowly relaxing component of a 15N-1H multiplet by the TROSY approach leads to substantial improvement in resolution; this is a prerequisite for accurate measurement of couplings from the 1H multiplets directly along the 1H frequency dimension or from the J-scaled doublets along the 15N frequency dimension.     

Optimization of Three-Dimensional TROSY-Type HCCH NMR Correlation of Aromatic H–C Groups in Proteins

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394–6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the ¹³C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S³ TPPI). The new 3D S³ TPPI TROSY HCCH method is demonstrated on a ¹³C,¹⁵N-labeled protein sample, RAP 18–112 (N-terminal domain of α₂-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.     

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.     

An α/β-HSQC-α/β Experiment for Spin-State Selective Editing of IS Cross Peaks

A generalized version of the TROSY experiment allows the spin-state selective editing of the four multiplet components of¹⁵N–¹H cross peaks of amide groups in proteins into four different subspectra, with no penalty in sensitivity. An improvement by in sensitivity results, if only two of the four multiplet components are selected. Use of the experiment for the measurement of¹J_HNcoupling constants is discussed. A water flip-back version of the experiment is demonstrated with a 45 kDa fragment of¹⁵N/²H labeledStaphylococcus aureusgyrase B.     

Spin-edited 2D HSQC-TOCSY experiments for the measurement of homonuclear and heteronuclear coupling constants: application to carbohydrates and peptides

Simple modifications of the sensitivity-improved HSQC-TOCSY pulse sequence are proposed for the easy determination of the sign and the magnitude of homonuclear and heteronuclear coupling constants. Whereas in well-resolved regions, a clean two-component E.COSY-like pattern allows a direct measurement from a single 2D spectrum, separate acquisition of equivalent single-component TROSY/anti-TROSY spectra becomes highly interesting when spectral crowding complicates the spectral analysis. It is also demonstrated that an additional restricted planar mixing element after the isotropic TOCSY process completely retains all spin-editing features and permits the accurate measurement of the sign and the size of the corresponding homonuclear proton-proton coupling constants. Among others, the proposed techniques are particularly suited for molecules presenting a great number of CH and NH spin systems. Examples and practical details of the implementation of these techniques on standard carbohydrates and peptides at 13C and 15N natural abundance are provided.     

Suppression of diagonal peaks in three-dimensional protein NMR TROSY-type HCCH correlation experiments

A novel method for suppression of (13)C-(13)C diagonal peaks without sensitivity loss in three-dimensional HCCH TROSY-type NMR correlation experiments involving aromatic side chains in proteins (Pervushin et al., J. Am. Chem. Soc. 120, 6394-6400 (1998)) is presented. The key element is a spin-state-selective filter in the (13)C-(13)C mixing sequence with the dual effect of selecting the TROSY resonance in the preceding evolution period and interchanging TROSY and anti-TROSY resonances. The cross peaks are invariant to this filter but diagonal peak coherence gets concentrated on the anti-TROSY transition so that it can be eliminated by a (13)C --> (1)H TROSY transfer element. The new method is demonstrated using a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz.     

Measurement of (1)J(NC') and (2)J(H(N))(C') couplings from spin-state-selective two-dimensional correlation spectrum.

A method for the measurement of (1)J(NC') and (2)J(H(N))(C') coupling constants from a simplified two-dimensional [(15)N, (1)H] correlation spectrum is presented. The multiplet components of the (1)J(NC') doublet in the indirect dimension and (2)J(H(N))(C') in the direct dimension are separated into two subspectra by spin-state-selective filters. Thus each subspectrum contains no more peaks than the conventional [(15)N, (1)H]-HSQC spectrum. Furthermore, the method for the measurement of (1)J(NC') and (2)J(H(N))(C') is designed to exploit destructive relaxation interference (TROSY). The results are verified against the measurements of (1)J(NC') from spin-state-selective [(13)C', (1)H] correlation spectra recorded with additional sequence described here.     

More line narrowing in TROSY by decoupling of long-range couplings: shift correlation and JNC′ coupling constant measurements

Since the introduction of RDCs in high-resolution NMR studies of macromolecules, there is a growing interest in the development of accurate, and sensitive methods for determining coupling constants. Most methods for extracting these couplings are based on the measurement of the splitting between multiplet components in J-coupled spectra. However, these methods are often unreliable since undesired multiple-bond couplings can considerably broaden the multiplet components and consequently make accurate determination of their position difficult. To demonstrate one approach to this problem, G-BIRD^(r) decoupled TROSY sequences are proposed for the measurement of ¹J_NH and ¹J_NC′ coupling constants. Resolved or unresolved splittings due to remote protons are removed by a G-BIRD^(r) module employed during t₁ and as a result, spectra with narrow, well-resolved peaks are obtained from which heteronuclear one-bond couplings can be accurately measured. Moreover, introduction of a spin-state-selective α/β-filter in the TROSY sequence allows the separation of the ¹J_NC′ doublet components into two subspectra which contain the same number of peaks as the regular TROSY spectrum. The ¹J_NC′ couplings are obtained from the displacement between the corresponding peaks in the subspectra.     

Simultaneous detection of resolved glutamate, glutamine, and gamma-aminobutyric acid at 4 T

A new approach is introduced to simultaneously detect resolved glutamate (Glu), glutamine (Gln), and gamma-aminobutyric acid (GABA) using a standard STEAM localization pulse sequence with the optimized sequence timing parameters. This approach exploits the dependence of the STEAM spectra of the strongly coupled spin systems of Glu, Gln, and GABA on the echo time TE and the mixing time TM at 4 T to find an optimized sequence parameter set, i.e., {TE, TM}, where the outer-wings of the Glu C4 multiplet resonances around 2.35 ppm, the Gln C4 multiplet resonances around 2.45 ppm, and the GABA C2 multiplet resonance around 2.28 ppm are significantly suppressed and the three resonances become virtual singlets simultaneously and thus resolved. Spectral simulation and optimization were conducted to find the optimized sequence parameters, and phantom and in vivo experiments (on normal human brains, one patient with traumatic brain injury, and one patient with brain tumor) were carried out for verification. The results have demonstrated that the Gln, Glu, and GABA signals at 2.2-2.5 ppm can be well resolved using a standard STEAM sequence with the optimized sequence timing parameters around {82 ms,48 ms} at 4 T, while the other main metabolites, such as N-acetyl aspartate (NAA), choline (tCho), and creatine (tCr), are still preserved in the same spectrum. The technique can be easily implemented and should prove to be a useful tool for the basic and clinical studies associated with metabolism of Glu, Gln, and/or GABA.     

Simple suppression of spurious peaks in TROSY experiments

In (1)H-(15)N TROSY experiments of proteins and nucleic acids, where the second coherence transfer delay time tau' has been fixed as 5.6 ms, 1/(2(1)J(NH)), in order to achieve complete spin-state selection, spurious negative peaks are observed along the (15)N axes. These peaks are often annoyingly large, especially for nucleic acids. A simple product operator calculation, however, indicated that the shortening of the second delay time tau', which is next to the t1 period, would efficiently suppress these spurious peaks, without sacrificing the sensitivities of the TROSY peaks too much. We have shown for three systems, two 11- and 17-kDa proteins and one 8-kDa DNA duplex, that these spurious peaks can be effectively suppressed with delay times of 3.3 ms for the two proteins and 2.3 ms for the DNA. These delay times, optimized by trial and error, for the spurious peak suppression did not depend on the magnetic field strength and the temperature very much. Although the shortened tau' delay times attenuate the TROSY peak intensities by about 10 and 20% for the two proteins and the DNA, respectively, this simple modification will be useful for the quantitative uses of TROSY peaks and will result in cleaner spectra for various TROSY-based multiple resonance experiments.     

Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times

Various strategies are described and compared for measurement of one-bond J(NH) and J(NC') splittings in larger proteins. In order to evaluate the inherent resolution obtainable in the various experiments, relaxation rates of (15)N-(1)H(N) coupled and heteronuclear decoupled resonances were measured at 600- and 800-MHz field strengths for both perdeuterated and protonated proteins. A comparison of decay rates for the two (15)N-?H(N)? doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz (1)H frequency, respectively, in the perdeuterated proteins. For the protonated proteins these ratios are 3.2 (800 MHz) and 2.4 (600 MHz). Relative to the regular HSQC experiment, the enhancement in TROSY (15)N resolution is 2.6 (perdeuterated; 800 MHz), 2.0 (perdeuterated; 600 MHz), 2.1 (protonated; 800 MHz), and 1.7 (protonated; 600 MHz). For the (1)H dimension, the upfield (1)H(N)-?(15)N? component on average relaxes slower than the downfield (1)H(N)-?(15)N? component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upfield (15)N-?(1)H? doublet component in slowly tumbling proteins makes it advantageous to derive the J(NH) splitting from the difference in frequency between the narrow downfield (15)N doublet component and either the (1)H-decoupled (15)N resonance or the peak position in an experiment which J-scales the frequency of the upfield doublet component but maintains some of the advantages of the TROSY experiment.     

MR imaging and cervical fixation devices: Evaluation of ferromagnetism,heating, and artifacts at 1.5 tesla

The purpose of this study was to assess ferromagnetism, heating, and artifacts for cervical fixation devices exposed to a 1.5 T MR system. Cervical fixation devices (three halos, one tong and two halo vests) were evaluated for compatibility with MR procedures. Ferromagnetism was determined using a previously described technique. Heating was evaluated by measuring temperatures at various positions on the cervical fixation devices while applied to a volunteer subject before and during the use of various pulse sequences, including an magnetization transfer contrast (MTC) sequence. Artifacts associated with routine clinical MR imaging of the cervical spine were qualitatively evaluated with the cervical fixation devices applied to a volunteer subject. None of the devices displayed attraction to the magnetic field. The temperature changes were ±1.5°C in each instance. The MTC pulse sequence produced a sensation of “heating” the skull pins that may have been caused by vibration of the cervical fixation device. The MR images of the cervical spine were obtained without apparent artifacts using each routine, clinical pulse sequence. The lack of ferromagnetism, negligible heating, and capability of obtaining diagnostically acceptable studies of the cervical spine indicate that MR imaging performed at 1.5 T or less may be conducted safely in patients with each of the cervical fixation devices tested using conventional pulse sequences.     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

Suppression of diagonal peaks in TROSY-type 1H NMR NOESY spectra of 15N-labeled proteins.

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of 15N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e. , large differences in 1HN linewidths depending on the spin state of attached 15N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated 15N-labeled protein sample, Neural Cell Adhesion Molecule 213-308 (NCAM) from rat, in H2O at 800 MHz.     

COSY with in-phase cross peaks

We have designed two pulse sequences that give rise to COSY-type spectra with in-phase multiplet structure. In these spectra, the cross peaks are absorptive and in-phase along both dimensions. Such 2D spectra are useful for quantitative measurements, such as measurement of the extent of H/D exchange in proteins and measurement of the concentrations of individual components in a series of mixtures. These spectra can also be used to emphasize cross peaks between weakly coupled protons.     

Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone.

A set of three improved two-dimensional (2D) NMR methods for measuring one-bond (15)N-(1)H coupling constants in the protein backbone is presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D spectra into two separate subspectra corresponding to the two possible spin states of the coupling partner. Cross talk between the two subspectra is a second order effect in the difference between the actual coupling constants and the one used in setting the pertinent delays of the pulse sequences. This relatively high degree of editing accuracy makes the methods useful for applications to molecules subjected to weak alignment where the one-bond coupling constants are linear combinations of a scalar J and a residual dipolar contribution containing important structural information. A demonstration of the new methods is shown for the (15)N-labeled protein chymotrypsin inhibitor 2 in a lipid bicelle mixture.     

Matter coupling and non-linear σ-models in N = 2 supergravity

Starting from an off-shell formulation of N = 2 supergravity, we construct the matter multiplet in its complex form (the scalar hypermultiplet) and in its real form. We construct the matter action which describes the coupling to supergravity. The interpretation where the 32 + 32 field components of the minimal gravitational multiplet are independent dynamical variables is known to be inconsistent for pure supergravity. We find it to be consistent when coupled to at least one matter multiplet, and to give rise to supersymmetric σ-models when coupled to at least two matter multiplets.