Abstract

The analysis of NMR spectra of med ium-size ( lo-20 kDa) proteins is often difficult because of severe signal overlap. A number of isotope-edited 2D experiments (I7) and, more recently, 3D NMR experiments have been used to alleviate this problem (8-12). Spreading spectral information in three independent frequency dimensions greatly reduces spectral overlap and thereby simplifies the process of analysis. The heteronuclear 3D experiments (I.?, 10-12) are particularly useful in this respect, because the total number of resonances remains unchanged relative to the corresponding homonuclear ‘H 2D spectrum; the chemical shift of the heteronucleus is merely used to disperse the resonances in the regular 2D spectrum along a third axis. All applications of heteronuclear 3D experiments to proteins publ ished to date combine the commonly used homonuclear ‘H experiments NOESY (14,15) and HOHAHA ( 16, 17) with the ‘H-detected heteronuclear mu ltiplequantum correlation (HMQC) experiment ( 18-20). Recently, it has been shown that the HMQC experiment does not provide as high a resolution as some more complex pulse sequences (21). However, the resolution in the dimension of the heteronuclear chemical shift is usually lim ited by digital resolution, and the potentially lower resolution does not play a role. So far, all protein applications of heteronuclear 3D NMR have utilized 15N as the heteronucleus. Here we report the first use of heteronuclear 3D protein NMR using the r3C chemical shift to spread the ‘H resonances. Although at first sight, the change from “N to 13C may appear simple, there were a number of problems that had made it uncertain whether this approach would be successful. F irst, the heteronuclear dipolar l ine-broadening effect of 13C on the resonance of its directly attached proton(s) is severe, typically causing a twofold increase of the regular proton linewidth. This sharply reduces sensitivity in those 3D experiments where one of the magnetization transfer steps relies on ‘H‘H Jcoupling. Our experiments with fully (about 95%) ‘3C-labeled protein also showed a reduction in the proton T, by about 30% relative to the unlabeled protein, and it was not certain a priori whether this strong heteronuclear dipolar relaxation would have a negative influence on the quality of the NOESY spectrum. A second problem arises from the presence of relatively large r Jcc couplings, varying from about 60 Hz for C,-carbonyl and aromatic couplings to about 35 Hz for couplings between aliphatic carbons. Attempting to resolve these splittings in the 3D experiment would increase the number of resonances and thus decrease signal-to-noise. As shown by Markley and co-workers ( 22-24)) using a relatively low level of 13C labeling reduces the * 3Cr3C broaden-

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