HCCH-TOCSY

Assignment of spin system TOCSY-TOCSY TOCSY-HMQC TOCSY-TOCSY 3D/4D HCCH-TOCSY HCCH-COSY HC(C)NH-TOCSY... 

Kay LE, Xu G-Y, Singer AU, Muhandiram DR, Forman-Kay JD. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 3H and 13C correlations in H20 samples of proteins. J Magn Reson 1993 101B 333-337. 

A more sensitive alternative is to use HCCH-TOCSY experiments [63-65], which transfer magnetization through the much larger 13C-13C coupling constants. In addition, these coupling constants are almost independent of the secondary structure and very uniform... 

The TROSY effect was also used in a relayed HCCH-COSY experiment to correlate adenine H2/H8 resonances in uniformly 13C-labeled RNA molecules [49], and significant sensitivity over the existing HCCH-TOCSY version was reported. Magnetization is transferred simultaneously in an out-and-back manner from H2 and H8 to the three aromatic carbon spins, C4, C5 and C6, establishing thus the connectivity within the adenine base spin system. 

In fully C-labeled proteins, the side chains of amino acids can be assigned using CCH or HCCH-TOCSY-type experiments (Fesik and Zuiderweg, 1990 Fesik et al., 1990 Bax et al., 1990b Kay et al., 1993). The three-dimensional HCACO-TOCSY experiment (Kay et al., 1992) is an extension of these experiments that uses the carbonyl chemical shifts to separate overlapping resonances. 

Sidechain H resonances can then be assigned from a 4D HCCH-TOCSY data set collected on the fully protonated protein sample. The Ca and Cp chemical shift values obtained directly from the protonated sample and the corrected C chemical shift values of the additional sidechain carbons should facilitate the analysis of the TOeSY data. 

Spectra were recorded on Bruker AMX-500 and DMX-750 NMR spectrometers at 30 C. All triple resonance experiments and the HCCH-TOCSY experiment were performed on a single sample of 15N, l3C-ubiquitin in 90% H2O/10% D20 buffer. HCCH-TOCSY spectra were obtained using the pulse sequence described by Bax et al. (1990) and utilized a 27 ms DIPSI-3 mixing sequence. The HCCH-TOCSY data sets were composed of 92 complex points in the... 

The essentially complete assignment of H, 13C and 15N resonances of recombinant human ubiquitin were obtained by analysis of HNCA, HN(CO)CA, HNCO and HCCH-TOCSY spectra and reference to previously reported H (Di Stefano and Wand, 1987) and 15n (Schneider et al., 1992) resonance assignments (Wand et al., 1996). Stereospecific assignments of leucine 5-CH3 and valine y-CHj groups were obtained using the isotopic labeling strategy developed by Neri et al. (1990). This approach provided unequivocal prochiral... 

Adiabatic pulses and isotropic mixing Use of adiabatic pulses for C,C and H,H TOCSY transfer, HCCH-TOCSY... 

Isotope edited experiments 3D HCCH-COSY 3D HCCH-TOCSY ID spin-state selective HMQC and HSQC-based 3D MUSIC CBCANH 3D MUSIC CBCA(CO)NH 2D Pro-HSQC... 

Clearly the homonuclear and the heteronuclear experiments could be combined in the reverse order i.e. HSQC-NOESY and HSQC-TOCSY. The main advantage in these schemes relates to N-edited experiments in which the narrower amide proton spectral width is sampled during f and the full proton spectral width is collected during t. On the other hand water suppression is more effective when HSQC follows NOESY. Also the sensitivity enhancement can be incorporated into the NOESY-HSQC experiment. Eor the TOCSY-HSQC or HSQC-TOCSY it does not matter because both the TOCSY and HSQC sequences can be implemented with the sensitivity enhancement. It should be mentioned that the TOCSY type of transfer is more effective between C nuclei than between protons and therefore the HCCH-TOCSY experiment is preferred when a doubly labeled sample is available. 

The first step in the conformational analysis of the oligosaccharide is the determination of complete proton and carbon resonance assignments. These have been reported previously for this particular trisaccharide [80], In systems with unknown assignments, the presence of uniform C-enrichment permits the application of conventional HCCH-COSY and HCCH-TOCSY experiments [81, 82], which invariably give complete resonance assignments in an efficient manner. 

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