1. Purpose

The HNCO experiment as described in Experiment 15.9 gives an unambiguous result by relating the HN proton resonance specifically to the CO carbon of the previous amino acid, thus providing inter-residue cross-peaks. The HN(CA)CO experiment shown here reveals mainly the connectivity to the CO carbon atom of the same amino acid (intra-residue cross-peak), using the 'J(N,C,) and 'J(C,,CO) spin coupling constants. In addition, the connectivity to the CO carbon of the preceding amino acid is often also seen by a transfer via 2(N,C,) and 'W(C,,CO). Thus, by comparing the spectra of both the HNCO and the HN(CA)CO methods, starting from one NH proton resonance, the signals of the CO ¹³C nuclei of the same and of the previous amino acid can be uniquely identified. Thus, by combining this with the HNCA and HN(CO)CA techniques, in theory one should be able to obtain complete sequencing information for the backbone.

Of the several variants known, we show here a gradient selected sequence, which has features very similar to the HN(CO)CA experiment 15.8, except that the C, and CO pulses have been interchanged. The sequence uses the echo/anti-echo scheme and the constant-time feature in the ¹⁵N dimension (F2), and States-TPPI in the ¹³C dimen- sion (F;), and is therefore phase-sensitive in all three dimensions. The sequence also provides a sensitivity enhancement by the preservation of equivalent pathways (PEP) principle, a Bloch–Siegert phase shift compensation, and a water flip-back pulse. The different pulses for the ¹³C,, and '°CO regions are generated by using band-selective pulses working at different offsets, with additional switching of the ¹³C transmitter offset. For this particular experiment we have chosen q-type Gaussian cascades. With 41 individual radiofrequency pulses, the sequence needs a considerable amount of preparation.

2. Literature

[1] R. T. Clubb, V. Thanabal, G. Wagner, J. Magn. Reson. 1992, 97, 213-217.

[2] J. Engelke, H. Riiterjans, J. Magn. Reson. Ser. B 1995, 109, 318-322.

[3] R. Bazzo, D. O. Cicero, G. Barbato, J. Magn. Reson. Ser. B 1996, 110, 65-68.

[4] J. Cavanagh, W. J. Fairbrother, A. G. Palmer III, N. J. Skelton, Protein NMR Spectroscopy, Academic Press, San Diego, 1996, 500-501.