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 ‘H, 13C, and “N resonance assignments of proteins uniformly enriched with “C and 15N. The new methods all rely on ‘H 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 ( l2 days) for protein concentrations on the order of I mM. One experiment (HNCO) correlates the amide ‘H and “N shifts with the 13C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ‘H and 15N shifts with the CLY 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 Ha and GY shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA( CO)N, correlates Ha and Cal resonances of one residue with the “N 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). o 1990Academic