Concerted two-dimensional NMR approaches to hydrogen-1, carbon-13, and nitrogen-15 resonance assignments in proteins.

When used in concert, one-bond carbon-carbon correlations, one-bond and multiple-bond proton-carbon correlations, and multiple-bond proton-nitrogen correlations, derived from two-dimensional (2D) NMR spectra of isotopically enriched proteins, provide a reliable method of assigning proton, carbon, and nitrogen resonances. In contrast to procedures that simply extend proton assignments to carbon or nitrogen resonances, this technique assigns proton, carbon, and nitrogen resonances coordinately on the basis of their integrated coupling networks. Redundant spin coupling pathways provide ways of resolving overlaps frequently encountered in homonuclear 1H 2D NMR spectra and facilitate the elucidation of complex proton spin systems. Carbon-carbon and proton-carbon couplings can be used to bridge the aromatic and aliphatic parts of proton spin systems; this avoids possible ambiguities that may result from the use of nuclear Overhauser effects to assign aromatic amino acid signals. The technique is illustrated for Anabaena 7120 flavodoxin and cytochrome c-553, both uniformly enriched with carbon-13 (26%) or nitrogen-15 (98%).