JOURNAL OF MAGNETIC RESONANCE 85,608-6 13 ( 1989) Solvent Suppression Using a Spin Lock in 2D and 3D NMR Spectroscopy with Hz0 Solutions BARBARAA.MESSERLE,GERHARDWIDER,GOTTFRIED OTTING, CHRISTOPHWEBER,ANDKURTW~~THRICH Institut fir Molekularbiologie und Biophysik, Eidgeniissische Technische Hochschule-Hiinggerberg, CH-8093 Zurich, Switzerland Received July 27, 1989 The ability to observe all polypeptide backbone amide protons in ’ H NMR spectra is a preliminary condition for the use of NMR to determine the structure of proteins in solution ( I ) . The acquisition of homonuclear 2D ‘H NMR spectra of proteins in HZ0 solution (2) was therefore a turning point in the methodological development which made it possible to obtain complete ‘H NMR assignments of proteins (3, 4)) and on this basis to determine three-dimensional protein structures (5). For proteins at slightly acidic pH, where the amide proton exchange rate is sufficiently slow, the most widely used solvent suppression method is by selective presaturation of the HZ0 line ( 1, 2,6). However, under certain conditions of pH and temperature, amide pro- tons may exchange sufficiently fast with the bulk water for their signal intensity to be significantly reduced on presaturation of H20. In addition, bleaching of spectral re- gions near the water line and concomitant loss of information in the two-dimensional spectra may also result from presaturation of the water resonance (2). As an altema- tive to solvent presaturation, one of many selective excitation methods ( 7-10) may be used to suppress the water signal. The main disadvantages of selective excitation techniques lie in the facts that they bring about phase distortions in 2D NMR spectra and that they cannot be combined with uniform spectral excitation across the entire spectrum. Today heteronuclear NMR experiments are increasingly used in protein structure determinations to provide supporting information for the resonance assign- ments and to facilitate the collection of the ‘H NMR data needed as input for the structure determination (11-13). In this paper we present a technique whereby the water signal can be suppressed in heteronuclear NMR experiments with proton detection, without the need for either water presaturation or selective excitation methods. It has been previously shown that suppression of undesired proton magnetization in heteronuclear spectra can be greatly enhanced by the use of spin-lock pulses ( 14). Here, spin-lock purge pulses are used during the application of heteronuclear pulse sequences to suppress the magnetization due to water protons. Figures IA and 1B show, respectively, the experimental schemes for 2D [ “N, ‘H] -COSY ( 1.5) and 3D NOESY- [ “N, ‘H ] COSY ( 12) supplemented by spin-lock purge pulses for solvent suppression. 0022-2364189 $3.00 . 608 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.