A number of heteronuclear 3D techniques have been de-constant-time period of 26-28 ms [2l / 2T(CO)] follows, veloped in recent years to obtain the complete assignment which allows optimum refocusing of the Ca, Cb coupling of 15 N, 13 C-labeled proteins (1). An experiment that often and also allows the buildup of the Ca, N coupling. At the proves useful is one that correlates backbone NH and N same time, the inclusion of a constant-time [2T(CO)] multiresonances with the intraresidue carbonyl resonance CO (2). ple-quantum period (Ca, CO) of 10-12 ms allows the moni-In the original experiment, named HN(CA)CO, the pulse toring of the directly attached carbonyl frequencies (t 1 ). scheme illustrated in Fig. 1a is used.
Finally, the coherence is transferred to the N resonances for Before discussing the improved performances of the new constant-time [2T(N)] monitoring (t 2 ) and then to the amide pulse sequence, we shall briefly outline the rationale of the protons for detection (t 3 ). The delay s of 1.78 ms, followed original experiment. NH proton magnetization is transferred by the proton inversion pulse, allows the refocusing of Ha, to N via an INEPT scheme (3). After a short delay for Ca couplings at the beginning of the sequence. The eliminarefocussing the NH, N coupling, a constant-time monitoring tion of this coupling term is necessary, since the coherence is of N resonance frequencies follows (t 1 ). During this period, finally transferred to a different nucleus (namely, the amide the coupling N-Ca builds up, thereby allowing the coherproton) for detection. For all residues except glycines, the ence transfer to Ca nuclei. After an additional constant delay transfer factor to the final observable term is sin (2pJ Ha,Ca s), for the evolution of the Ca, CO coupling, the magnetization which is equal to one for J Ha,Ca ร
140 Hz. For glycine is finally transferred to the carbonyl nuclei for monitoring residues, the coupling of Ca coherences cannot be efficiently (t 2 ). Then, by reversing the pathway just described, the magrefocused with respect to both a protons at the same time, netization is brought back to the protons whence it originally and the transfer factor is sin(2pJ Ha,Ca s)cos(2pJ Ha,Ca s), started for detection (t 3 ). In this pulse sequence, two relawhich is equal to zero. Therefore, as a direct consequence of tively long delays (each about 25 ms) are needed in order the coherence-transfer pathway of this experiment, glycine to transfer the magnetization from N nuclei to Ca nuclei signals are expected to be filtered out or at least severely and vice versa (J N-Ca ร
11 Hz). During these delays, the diminished, depending on the actual value of the coupling magnetization resides on the N nuclei. Moreover, two addiconstant J Ha,Ca involved (4). The delay r of 2.7 ms allows tional delays (each about 7 ms) are used for the coherence the build up of the N, NH couplings prior to the final magneto be transferred to the carbonyl nuclei from the Ca nuclei tization transfer to amide protons for detection. and vice versa (J Ca -CO ร
55 Hz). During these periods, the We named our pulse sequence (HACA)CO,NH where magnetization resides on the Ca nuclei. In the center of the the nuclei whose frequencies are monitored are outside pulse sequence, the incremented t 2 period is used to monitor brackets. The new pulse scheme (Fig. 1b) provides better the carbonyl frequencies.
sensitivity, thanks to several factors. First, the total duration In Fig. 1b, we illustrate an alternative pulse scheme, which of the pulse sequence is about 30 ms shorter than the original allows better sensitivity to be obtained. The magnetization one. However, the advantage, in terms of diminished relaxsources are the Ha nuclei, whose coherence is transferred ation loss, is partially reduced by the increased residence to the directly attached Ca nuclei via an INEPT scheme. A time of the magnetization on the usually fast relaxing Ca nuclei (10-12 ms as multiple-quantum coherence out of 26-28 ms in total, as opposed to 14 ms in the original