3-DIMENSIONAL TRIPLE-RESONANCE NMR-SPECTROSCOPY OF ISOTOPICALLY ENRICHED PROTEINS

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 (1-2 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 Ca chemical shift. This experiment often also provides a weak correlation between the amide NH and 15N resonances of one amino acid and the Cα 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 Hα and Cα 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). © 1990.