Water proton spin saturation affects measured protein backbone 15N spin relaxation rates

摘要

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 Escherichia coli. 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) 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.