Journal of Biomolecular NMR, 2 (1992) 395-400 395 ESCOM J-Bio NMR 074 Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques Adrian L. Davis, Rolf Boelens and Robert Kaptein* Department of Chemistry, Bijvoet Centrefor Biomolecular Research, University of Utrecht, Padualaan 8. 3584 CH Utrecht, The Netherlands Received 11 May 1992 Accepted 5 June 1992 Keywords." Triple-resonance; Pulsed fieldgradients; 3D NMR; Proteins SUMMARY A rapid method for recording three-dimensional triple-resonance experinaents utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein. A number of 3D triple-resonance experiments which allow the sequential assignments of backbone resonance in uniformly ~3C, ~SN-labeled proteins have recently been proposed (Kay et al., 1990). One such experiment is the HNCO experiment, which correlates the amide IH and ISN resonances of an amino acid with the carbonyl resonance of the preceding residue. Such sequen- tial connectivities are combined with the intra-residue and sequential information obtained from HCACO and HCA(CO)N experiments (Ikura et al., 1990) or from HN(CA)CO (Clubb et al., 1992), and in conjunction with other experiments lead to a complete characterisation of the backbone resonances. The HNCO experiment is very sensitive, as the magnetisation transfers are mediated by the large tJNH and UNc' scalar couplings, and hence it is desirable to record the 3D spectrum with the minimum number of transients per (t~:2) increment. The use of pulsed field gradient techniques (Maudsley et al., 1978; Bax et al., 1980; Barker and Freeman, 1985) to select the desired coherence transfer pathways has undergone a recent resur- gence of interest (H urd, 1990; Davis et al., 1991 ; Vuister et al., 1991 ) as a result of improvements in the design of gradient coils, and offers the tantalising prospect of recording 3D experiments *To whom correspondence should be addressed. 0925-2738,:$5.00 ~ 1992ESCOM SciencePublishers B.V.