HCCH-TOCSY experiments

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... 

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... 

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. 

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. 

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... 

BIOLOGICAL MACROMOLECULE STRUCTURAL INFORMATION HCCH-COSY and -TOCSY Experiments... 

The assignment of side chain resonances is a more laborious undertaking. The proton detected versions of the aforementioned experiments such as HN(CA)HA and HN(CAGE) HAHB reveal the protons at the stem of the side chain. Customarily carbons and aliphatic protons are obtained by CC(CO)NH, HC(C-CO)NH, HCCH-COSY and -TOCSY experiments. Furthermore there are special experiments to correlate nuclei in the aromatic and polar side chains. 

Figure 18. Pulse sequence diagrams for 3D H(CA)P-CC-TOCSY (a) and HCCH (b) experiments. (Reproducedfrom reference 19. Copyright 2001 American Chemical Society.)...

To overcome the problem of low dispersion of sugar resonances in nucleotides Hu et al. proposed to incorporate HCN transfer into HCCH-type sequences resulting in 3D MQ-HCN-CCH-TOCSY and 3D MQ-HCN-CCH-COSY experiments. The coherence transfer pathway starts with HT-CT transfer followed by an out-and-back transfer to N1/N9 for indirect detection and subsequent transfer through network. As a result the CCH spectra are additionally resolved with chemical shift. During the C -N1/N9 transfer MQ HT/CT coherence is active to reduce the relaxation losses due to relaxation. The experiments were successfully applied to a 23-mer RNA aptamer. 

Multidimensional correlation experiments can be arduous to analyse at the best of times. Therefore, any practitioner ofbiomolecularNMR spectroscopy will want to do the minimum number of experiments to achieve unique and unambiguous resonance assignment of as many amino-acid residue nuclei as necessary in order to enable the critical NOESY experiments. Two of the simplest 3D correlation experiments that have been used are 3D //- NTOCSY-HSQC and 3D HCCH COSY/TOCSY. Such 3D correlation experiments are known as doubleresonance experiments in that they generate intensity data /(Fi, Fj, F3) emanating from the double resonance of two entirely different populations of nuclei, either Hand Nnuclei, or H and C nuclei respectively. 

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