On the accurate measurement of amide one-bond 15N-1H couplings in proteins: effects of cross-correlated relaxation, selective pulses and dynamic frequency shifts.
Amide one-bond 15N-1H scalar couplings of 15N- and [15N,2H]-isotopically enriched ubiquitin have been measured with the Quantitative J approach by monitoring NMR signal intensity modulation. Scalar couplings of the non-deuterated protein are in average approximately 0.6 Hz larger than values of deuterated ubiquitin. This deviation is 30 times the error derived from experiment reproducibility. Refocusing dipole/dipole cross-correlated relaxation decreases the discrepancy to approximately 0.1 Hz, suggesting that it likely originates from relaxation interference. Alternatively, the subtraction of J values obtained at different magnetic fields largely reduces the relaxation effects. In contrast, the dynamic frequency shift whose main contribution to 1J(15N-1H) arises from 15N chemical shielding anisotropy/NH dipole cross-correlation, is not eliminated by refocusing spin evolution under this interaction. Furthermore, the average difference of 1J(15N-1H) values at two magnetic fields closely agrees with the theoretical expected difference in the dynamic frequency shift.
de Alba
Eva
E
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA. dealbae@nhlbi.nih.gov
Tjandra
Nico
N
D000465:Algorithms; D000577:Amides / Q000032:analysis* / Q000737:chemistry*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D011522:Protons; D011846:Radio Waves; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted
D000577:Amides; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D006859:Hydrogen