Search

NOESY NMR spectra provide interproton distance information for a molecule in solution and the complete, unambiguous determination of NOESY spectral assignments is the basis for protein structure determination. High resolution NOESY can be obtained from 13 C and 15 N isotope edited four-dimensional (4D) data, but these experiments would normally require weeks to complete. We have applied a G-matrix Fourier transform and time-sharing (GFT-TS) NMR method for simultaneously acquiring two sets of 4D NOESY data. The implementation of the GFT-TS allows 2.5- to 5-fold reduction in experimental time without sacrificing spectral resolution as compared with that of 3D data. The 13 C, 15 N-edited GFT-TS (4,3)D H – N – CN –H NOESY (GFT dimensions are underlined and TS dimensions are in italics) provides convenient and unambiguous NOE assignments for HN/HN and HN/HC for a sample of 1.4 mM ubiquitin (76 amino acids, 8.5 kDa). We also provide a set of utility scripts for data processing and spectral assignment to facilitate the use of GFT NMR. This method shows great promise for routine high quality NMR NOESY data collection for small to medium sized proteins.

The J-multiplied HSQC experiment (MJ-HSQC: S. Heikkinen et al., J. Magn. Reson 137, 243 (1999)) amplifies J coupling constants m times and allows direct observation of the (3)J(HNHalpha) coupling constants of peptides and proteins (10 kDa). The drawbacks to this method are line broadening in the f(1)-dimension and lower sensitivity. In the J-multiplied HMQC (MJ-HMQC) experiment described here, the PEP-HSQC pulse sequence is replaced by a sensitivity-enhanced HMQC section, and the total decay time for the J-coupling and the chemical shift evolution is shortened by a period of t(1). This experiment affords narrower linewidth and enhances the sensitivity by 34%, on an average of 105 well-isolated peaks, when compared with the MJ-HSQC experiment.

Previous theoretical calculations have demonstrated that the multiquantum relaxation rate of (1)H(alpha)-(13)C(alpha)(R(MQ)) is, on average, 1.3 +/- 0.4 or 1.7 +/- 0.6 times slower than the single-quantum relaxation rate of (13)C(alpha)(R(C)) for a sample with or without, respectively, amide protons. By taking advantage of this fact and by using the PEP sensitivity enhancement scheme, an HMQC version of the HCACO experiment has been developed. We demonstrate that this new experiment is 23 and 55% more sensitive than the original HSQC version of the HCACO experiment, at constant times of 7 and 27 ms, respectively, for a sample of the BC domain of the ciliary neurotrophic factor receptor protein dissolved in D(2)O at 20 degrees C.