Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination.

A procedure is described that affords complete 1H, 13C and 15N resonance assignment in proteins of up to about 25 kDa. The new approach requires uniform isotopic enrichment of the protein with 13C and 15N and correlates resonances of adjacent nuclei using the relatively large and well-resolved one-bond J couplings. Spectral overlap, a common problem in the application of two-dimensional NMR, is removed by increasing the dimensionality of the new methods to three or four, without increasing the number of observed resonances. With complete 1H, 13C and 15N resonance assignments available, the nuclear Overhauser effect (NOE)-based interproton distance constraints can be extracted in a very straightforward manner from four-dimensional NOE spectra.