4-DIMENSIONAL C-13/C-13-EDITED NUCLEAR OVERHAUSER ENHANCEMENT SPECTROSCOPY OF A PROTEIN IN SOLUTION - APPLICATION TO INTERLEUKIN 1-BETA

A four-dimensional C-13/C-13-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (> 95%) C-13-labeled interleukin 1-beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the C-13 chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the C-13 chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different C-13 chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another. The 4D C-13/C-13-edited NOESY experiment is characterized by high sensitivity as the through-bond transfer steps involve the large J1CH (130 Hz) couplings, and it is possible to obtain high-quality spectra on 1-2 mM samples of C-13-labeled protein in as little as 3 days. This experiment should open up the application of protein structure determination by NMR to a large number of medium-sized proteins (150-300 residues) of biological interest.