Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990

Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backbone ¹H, ¹³C, and ¹⁵N resonance assignments of proteins uniformly enriched with ¹³C and ¹⁵N. The new methods all rely on ¹H detection and use multiple magnetization transfers through well-resolved one-bond J couplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2 days) for protein concentrations on the order of 1 mM. One experiment (HNCO) correlates the amide ¹H and ¹⁵N shifts with the ¹³C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ¹H and ¹⁵N shifts with the Cα chemical shift. This experiment often also provides a weak correlation between the amide NH and ¹⁵N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Hα and Cα shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with the ¹⁵N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7 kDa).