HCCH-COSY

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

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. 

Assignment of protons in side chains is also critical HCCH-COSY and HCCH-HOHAHA are widely used to facilitate these assignments. Because each amino acid has an asymmetric center, stereospecific assignments for methylene and methyl protons are obtained whenever feasible. 

A markedly increased bandwidth of heteronuclear Hartmann-Hahn transfer for a given average rf power can be achieved with the MGS-1 and MGS-2 sequences developed by Schwendinger et al. (1994) (see Fig. 33G and H). The sequences are MLEV-4 and MLEV-8 expansions of new composite pulses R, which consist of square pulses with rf phases of 0 or 180° and different rf amplitudes that are separated by delays (see Fig. 34). Figure 35 shows HCCH-COSY spectra of a fully C-labeled protein using DIPSI-2 and MGS-2 for the initial polarization transfer from H to (prepolarization) as well as for back-transfer from to H (Majumdar et al., 1993). Note that the absolute bandwidth of MGS-2 is markedly increased compared to DIPSI-2, even though the average power... 

Fig. 35. Experimental HCCH-COSY spectra of the fully C-labeled protein rhodniin with two heteronuclear Hartmann-Hahn transfer steps. The spectra were acquired at a spectrometer frequency of 600 MHz employing DIPST2 with = 4.8 kHz (A) and MGS-2 with = 3.78 kHz (B). In the experiments the heteronuclear Hartmann-Hahn mixing periods had a duration of 6 ms. The same plot levels were used for both spectra. (Adapted from Schwendinger et al., 1994, courtesy of Academic Press.)...

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... 

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. 

By comparison, 3D HCCH COSY experiments are a little simpler (Figure 5.22). An initial pulse develops transverse magnetisation in aliphatic H nuclei (specifically C - H atoms) (source nuclei I) (Fi) that evolves according to chemical shift before polarisation transfer... 

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. 

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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