Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins
Publication year: 2011
Source: Journal of Magnetic Resonance, Volume 213, Issue 2, December 2011, Pages 423-441
Lewis E.*Kay, Mitsuhiko*Ikura, Rolf*Tschudin, Ad*Bax
Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backboneH,C, andN resonance assignments of proteins uniformly enriched withC andN. The new methods all rely onH detection and use multiple magnetization transfers through well-resolved one-bondJcouplings. 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 amideH andN shifts with theC shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amideH andN shifts with the C?chemical shift. This experiment often also provides a weak correlation between the amide NH andN resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the H?and C?shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with theN 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 undesiredJsplittings 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).
Source:
Journal of Magnetic Resonance