Optimization of TROSY- and anti-TROSY-based 15N CPMG relaxation dispersion experiments through phase cycling

CPMG relaxation dispersion studies of biomolecular dynamics on the μs–ms timescale can provide detailed kinetic, thermodynamic, and structural insights into function. Frequently, the ¹⁵N spin serves as the probe of choice, as uniform incorporation of the ¹⁵N isotope is facile and cost-effective, and the interpretation of the resulting data is often relatively straightforward. In conventional CPMG relaxation dispersion experiments the application of CPMG pulses with constant radiofrequency (RF) phase can lead to artifactual dispersion profiles that result from off-resonance effects, RF field inhomogeneity, and pulse miscalibration. The development of CPMG experiments with the [0013]-phase cycle has significantly reduced the impact of pulse imperfections over a greater bandwidth of frequency offsets in comparison to constant phase experiments. Application of ¹⁵N-TROSY-based CPMG schemes to studies of the dynamics of large molecules is necessary for high sensitivity, yet the correct incorporation of the [0013]-phase cycle is non-trivial. Here we present TROSY- and anti-TROSY-based ¹⁵N CPMG experiments with the [0013]-phase cycling scheme and demonstrate, through comprehensive numerical simulations and experimental validation, enhanced resistance to pulse imperfections relative to traditional schemes utilizing constant phase CPMG pulses. Notably, exchange parameters derived from the new experiments are in good agreement with those obtained using other, more established, ¹⁵N-based CPMG approaches.