CT-HSQC

D 1H-13C CT-HSQC/HMQC identifies proton-carbon spin pairs in both sugar and base. The constant time evolution removes the peak splitting due to 13C-13C coupling. The proton and carbon resonances are subsequently connected by... 

Conceptually similar to FIDS is the so-called /-modulated CT-HSQC experiment [14] (Fig. 7.8b). The coupling evolves in one experiment during x and in a second experiment not at all. The intensity ratio between first and second experiment is cos (njx). As an example we show the measurement of an NH dipolar coupling by this method. The pulse sequence of the constant time HSQC and the oscillatory behavior of the cross-peak intensities are shown in Fig. 7.11. 

Figure 25 Five himdred megahertz H, ct-HSQC spectra (aromatic region) of the hairpin ribozyme B domain, 30 °C, withont (a) and with (b) the addition of 20 aM MnCl2. Resonances that are broadened as a result of Mn binding are labeled with their corresponding numbers in the spectrum without Mn +. (Adapted from Butcher, Allain and Feigon. 2000 American Chemical Society)...

Fig. 1. H- - C CT-HSQC spectrum of a sample of 1.5mM Val, Leu, lie (51) methyl-protonated maltose-binding protein (MBP), 2mM /3-cyclodextrin, 20 mM sodium phosphate (pH 7.2), 3mM NaN(, 200pM EDTA, 0. 1 mg/ml Pefabloc, 1 /ig//d pepstatin and 10% D20 recorded at 37°C, on a Varian Unity-1- 500-MHz spectrometer. Acquisition times of 28 and 64 ms were employed (/, t2) along with a relaxation delay of 1.5 s, fora total measuring time of 3 h. (a) Aliphatic region of the H- - C correlation map of MBP, illustrating the selectivity of labelling. Small amounts of residual protonation are observed at the Cy positions of a number of Pro/Arg residues, the Cp positions of Asp and Ser (aliased) residues, and the Cy2 methyl positions of lie. In all cases, intensities of these cross-peaks are less than 10% of the methyl peaks, (b) Methyl region of the H- - C HSQC. Reproduced with permission from Kluwer Academic Publishers Goto et al.H...

Fig. 36. 2D- Cy " J-Spin-echo-difference H/ N-CT-HSQC spectra. Only residues with large couplings give signals in the spectrum indicating an antiperiplanar conformation...

CT-HSQC)—a new NMR method to measure accurate one-bond J and RDCs with strong couplings in natural abundance, J. Magn. Reson. 228 (2013) 159—165. 

In addition to sensitivity-improved two-dimensional TOCSY experiments, PEP versions of two-dimensional HSQC-TOCSY experiments (Cavanagh et al., 1991) as well as three-dimensional HSQC-TOCSY and three-dimensional TOCSY-HMQC experiments (Palmer et al., 1992 Ranee, 1994 Krishnamurthy, 1995) have been reported. This enhancement scheme is also used in heteronuclear coherence-order-selective coherence transfer (COS-CT Schleucher et al., 1994 Sattler et al., 1995a). Because in... 

To obtain information about the glycosidic linkage, 1H—1H NOESY/ROESY and/or long-range 1H—13C correlated spectra, heteronuclear multiple bond correlation (HMBC) or CT (constant time)-HMBC,12 are recorded. The combined 2D HSQC(HMQC)-NOESY(ROESY) experiments could also be helpful, but have limited applications due to their low sensitivity in samples with natural abundance of 13C. 

Intensity-based methods. 1H— H RDCs have been obtained by analyzing the intensity ratios of the diagonal and cross-peaks in a series of 2D CT COSY spectra.177 This method can only be applied to resolved resonances, for example, those of anomeric protons.149 Similar limitations apply to /-modulated ID directed COSY,153,178 which uses selective 180° pulses to produce a series of ID spectra for each pair of coupled spins. This approach has recently been extended to include additional selection blocks yielding a versatile method for the measurement of coupling constants in compounds with severely overlapping proton resonances such as those found in carbohydrates.154 The problem of overlapping resonances can also be resolved by involving 13C nuclei, as demonstrated on natural abundance (13C COSMO HSQC)32 or uniformly 13C isotopically enriched carbohydrates (2D-HSQC-(sel C, sel H)-CT COSY experiment).73,158... 

The readout of line frequencies is simpler in Fb but potentially less precise due to limited digital resolution. The digital resolution can be increased if long-range, either proton-carbon or proton—proton, interactions are removed by the action of a BIRD pulse.36,175,180 If required, overlap reduction can be achieved by separating the a//3 states into two spectra as demonstrated in S3-CT-HSQC37,147,148 or SPITZE (spin state selective zero overlap)-HSQC.170... 

A new element for simultaneous indirect detection of C and signals in labelled proteins was proposed by Uhrin et The CT- CHs, VT- N-HSQC sequence combines constant-time carbon evolution with variable delay nitrogen evolution. This is achieved by the variation of the position of a 90° pulse creating transverse coherence within the C constant-time period. The maximum indirect acquisition time for both nuclei is determined by constant-time period set to l/ /(C,C) = 28.6 ms. The method is best suited for detection of CH3 signals due to their slower relaxation. The proposed element was incorporated into NOESY-based experiments resulting in 3D NOESY-CH3NH and 3D HSQC-NOESY-CH3NH sequences. The experiments... 

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly C istopically enriched samples (U- C) of oligosaccharides or oligonucleotides, has been proposed by Martin-Pastor et al for the measurement of proton-proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, C- C CT COSY, has been proposed for the measurement of one bond carbon-carbon RDC in these molecules. 

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