It has long been recognized that stereospecific reso-angles in asparagine and glutamine side chains, respectively, and it may not always be obvious which resonance nance assignments significantly improve the quality of protein structures determined by NMR ( 1 -8 ) . Both the belongs to which position. Especially in larger proteins, the analysis of intraresidual NOE intensities can be ren-number and the precision of distance constraints involving diastereotopic methylene protons and isopropyl dered difficult by spin diffusion and spectral overlap. In this Communication, a sensitive two-dimensional method methyl protons is enhanced when this kind of information is available. Without taking recourse to long-range dipo-is described which exploits vicinal 1 H, 13 C couplings in order to differentiate between the two positions. lar interactions in known tertiary structures, these assignments can be obtained from a combination of vicinal cou-
The pulse scheme depicted in Fig. 1 is based on the HNCO experiment ( 28 ) , employing an HSQC-rather pling constants and intraresidual NOEs. The most reliable strategy makes use of the incorporation of stereoselec-than an HMQC-type transfer between 15 N and 13 CO spins.
Since 15 N antiphase magnetization with respect to the tively deuterated amino acids ( 9 -14 ) or biosynthetically directed fractional 13 C labeling ( 15 -18 ) . A similar ap-attached protons is not refocused, correlations of the sidechain amide groups can be detected with the same inten-proach is, of course, not applicable for the individual assignment of exchanging nuclei such as side-chain am-sity as those of the polypeptide backbone. In uniformly 13 C-labeled proteins, the evolution of the one-bond cou-ide protons of asparagine and glutamine residues. Although methods have been introduced for the selective pling between the carbonyls and the directly bound aliphatic carbons leads to a splitting of the cross peaks in detection of NH 2 groups ( 19, 20 ) , no distinction can be made for signals of protons in the trans ( H d21 and H e21 the F 1 dimension. Pulses on 13 CO are applied band-selectively in order not to disturb the spin states of the aliphatic for Asn and Gln, respectively ) and cis ( H d22 and H e22 ) carbons. As a result, E.COSY-like ( 29 ) multiplets are configuration with respect to the carbonyl oxygen.
obtained from which 3 J ( H d , C b ) and 3 J ( H e , C g ) coupling In random conformations, a chemical-shift difference constants can be determined for asparagine and glutamine of approximately 0.7 ppm was reported for the two gemiside chains, respectively. Solvent suppression is achieved nal protons ( 21) . The resonance frequencies for the trans with the WATERGATE ( 30 ) technique, while the saturaprotons of primary amide groups in small organic comtion of the water magnetization is avoided by keeping it pounds ( 22) and Asn and Gln derivatives ( 23) , as well along the z axis during the application of B 0 -gradient as in Asn-containing peptides ( 24 ) , were correspondingly pulses with the use of water-selective 90Њ pulses ( 31, 32 ) . found to appear to low field from those of the cis protons,
The final 15 N pulse is displaced from the center of the but in proteins this order can be reversed by the influence reverse INEPT delay ( 33 ) , allowing the use of longer of aromatic rings in the vicinity of the NH 2 groups or by and thus more selective pulses on the H 2 O resonance, hydrogen-bond formation. In some cases, the individual while fixing the duration for refocusing of proton antiassignment can be obtained on the basis of intraresidual phase magnetization at ( 2 1 J NH ) 01 Å 2t. ( i.e., H b -H d , H g -H e ) NOE contacts ( 25 -27 ) as sug-
The H 2 ( N ) CO -E.COSY sequence was applied to the gested by Montelione et al. ( 24 ) . The intensity ratio beindividual assignment of side-chain amide resonances of tween the intraresidual NOE cross peaks of the trans and 13 C / 15 N-labeled flavodoxin from the sulfate-reducing orcis protons, however, depends on the x 2 and x 3 torsion ganism Desulfovibrio vulgaris. The protein, which consists of 147 amino acids and a noncovalently bound FMN molecule, was dissolved in 10 m M potassium phosphate * To whom correspondence should be addressed.