Water proton spin saturation affects measured protein backbone 15N spin relaxation rates.
Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios (s/n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1HN recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (<S2>=0.81) than the non-saturated ones (<S2>=0.88), indicating such order parameters may be previously underestimated.
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, United States.
Kang
K
Chen
Nico
N
Tjandra
D000465:Algorithms; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry; D016880:Anisotropy; D016477:Artifacts; D018076:DNA, Complementary / Q000737:chemistry; D056324:Diffusion Tensor Imaging; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen / Q000737:chemistry*; D009587:Nitrogen Isotopes; D011522:Protons; D059629:Signal-To-Noise Ratio; D014867:Water / Q000737:chemistry*
D026921:Amino Acid Transport Systems, Neutral; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011522:Protons; C528486:glnH protein, E coli; D014867:Water; D009584:Nitrogen