TROSY-HNCO

Fig. 4. The HNCO-TROSY experiment for recording solely interresidual 1HN, 15N, 13C correlations in 13C/15N/2H labelled proteins. All 90° (180°) pulses for the 13C and 13C spins are applied with a strength of 2/ /l5 (p/ /3), where 2 is the frequency difference between the centres of the 13C and 13Ca regions. All 13Ca pulses are applied off-resonance with phase modulation by Q. A = 1/(4/hn) Tn = l/(4/NC ) S = gradient + field recovery delay 0 < k < TN/z2,max- Phase cycling i = y 4>2 = x, — x + States-TPPI 03 = x 0rec = x, — x.

The magnetization has now been successfully transferred from the HN spin to the intraresidual and sequential 13C spins or alternatively to the interresidue 13C spin either using HNCA-TROSY or HNCO-TROSY schemes, respectively. It is inevitable that the HNCO-TROSY spectrum cannot be used for the sequential assignment alone because it does not bridge two sequential N shifts through common carbonyl carbon frequency. The... 

In the alternative approach, the HN(i), 15N( j, 13C (i/i— 1) correlations in the HNCA-TROSY spectrum can be supplemented with the data from the HN(CO)CA-TROSY experiment72 73 yielding solely 11 IN(/), 15N( ), 13C (i- 1) correlations. To this end, the HNCO-TROSY experiment is extended with the 13C —> 13C INEPT step, which utilizes rather large (ca. 51-55 Hz) one-bond scalar coupling between the 13C and 13C spins in order to transfer magnetization from the 13C (< — 1) nucleus further to the 13C ( — 1) spin. 

Fig. 23. Coherence transfer efficiency as a function of delay 2Tn f°r the HNCO-TROSY scheme. Following parameters were used [Eq. (12)] T 2,i5n = 50 ms, VNc = 15 Hz.

HNfz) connectivities with good sensitivity, excluding serines, threonines, leucines, and glycines (and probably valines and prolines). Thus, the use of HNCO-TROSY/HN(CA)CO-TROSY experiment pair can be very useful in assigning high molecular weight proteins. 

D HCN-based 2D HNCO-TROSY-based 2D HN(CO)CA-TROSY-based 2D HNCA-TROSY-based 2D HNCO-based... 

The HNCA-TROSY experiment can be readily extended to correlate the 13C spin of the preceding residue with intraresidual 11 IN(/), 15N(i), and 13C° (/ ) frequencies. This kind of four-dimensional HNCO CA-TROSY experiment (Fig. 7) was recently introduced by Konrat et al.79 The coherence flows through the following pathway... 

The set of four-dimensional (HNCO, iCA, HNCOCA and HNCACO) TROSY experiments has been successfully applied in the assignment of several large proteins.12,25,26 These three experiments are able to resolve... 

Residual dipolar couplings 3D TROSY-HNCO-based... 

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