Abstract
Nuclear magnetic resonance relaxation dispersion (rd) experiments provide kinetics and thermodynamics information of molecules undergoing conformational exchange. Rd experiments often use a Carr-Purcell-Meiboom-Gill (CPMG) pulse train equally separated by a spin-state selective inversion element (U-element). Even with measurement parameters carefully set, however, parts of 1H–15N correlations sometimes exhibit large artifacts that may hamper the subsequent analyses. We analyzed such artifacts with a combination of NMR measurements and simulation. We found that particularly the lowest CPMG frequency (νcpmg) can also introduce large artifacts into amide 1H–15N and aromatic 1H–13C correlations whose 15N/13C resonances are very close to the carrier frequencies. The simulation showed that the off-resonance effects and miscalibration of the CPMG π pulses generate artifact maxima at resonance offsets of even and odd multiples of νcpmg, respectively. We demonstrate that a method once introduced into the rd experiments for molecules having residual dipolar coupling significantly reduces artifacts. In the method the 15N/13C π pulse phase in the U-element is chosen between x and y. We show that the correctly adjusted sequence is tolerant to miscalibration of the CPMG π pulse power as large as ±10% for most amide 15N and aromatic 13C resonances of proteins.
Highlights
Relaxation dispersion experiments of nuclear magnetic resonance (NMR) are one of the most commonly used methods to obtain valuable information about the kinetics and thermodynamics of molecules for which the conformations exchange between those in the major-populated visible state and in the minor-populated invisible state or among more conformational sub-states [1,2,3,4]
When the 15N π pulse of the U-element was applied along the axis shifted by 90◦ (i.e., x), large artificial R2eff values were found as shown in the blue profile in Figure 2a, at the lowest νcpmg value (25 Hz), where a single 15N spin-echo was placed within each CPMG period (δ = 10 ms, the total relaxation time, 4*δ, = 40 ms)
The pulse sequence accumulated imperfections related to the CPMG π pulses, including the off-resonance effects and power miscalibration, on the transverse relaxation optimized spectroscopy (TROSY) magnetization component without canceling imperfections during the paired CPMG periods
Summary
Relaxation dispersion (rd) experiments of nuclear magnetic resonance (NMR) are one of the most commonly used methods to obtain valuable information about the kinetics and thermodynamics of molecules for which the conformations exchange between those in the major-populated visible state and in the minor-populated invisible state or among more conformational sub-states [1,2,3,4]. In rc heteronuclear transverse relaxation optimized spectroscopy (TROSY)-based CPMG experiments [18], a false large R2eff is often observed, at low repetition rates of the CPMG refocusing π pulses, even when the radio frequency (rf) magnetic field strength of the 15N or 13C π pulses is accurately calibrated. Our simulation showed that the off-resonance effect and pulse miscalibration generated artifacts at distinct off-resonance positions This method is immediately and implementable in a wide range of rc CPMG sequences, but it must be noted that the correct phase of the central π pulse in the U- or P-element is different depending on the spectrometer designs. The above-mentioned mechanisms are illustrated by a vector model, which may explain them more and intuitively than the average Hamiltonian-based calculation
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