NMR Relaxation Dispersion Experiments

Backbone amide ¹⁵N CPMG relaxation dispersion experiments were performed at static magnetic field strengths of 11.7 T, using a Varian/Agilent VNMRS Direct Drive spectrometer equipped with a room-temperature triple-resonance probe, and 14.1 T, using a Bruker Avance NEO spectrometer equipped with a 5 mm HPCN QCI cryoprobe. All experiments were performed at a temperature of 301 K. Temperature calibration was performed prior to each series of relaxation experiments using a neat protonated methanol sample.^32,33 The sample pH was adjusted immediately before each series of relaxation experiments and checked after each series to ensure that the sample pH had not drifted. Constant time relaxation-compensated CPMG experiments^34,35 were performed at 11.7 T using a 40 ms constant time relaxation period with CPMG refocusing frequencies, ν_cpmg, of (3 × 0, 50, 100, 150, 200, 300, 400, 500, 650, 800, and 950) Hz and interleaved sampling of t₁ points with different values of ν_cpmg. Experiments were acquired with a 2 s recovery delay, 80 scans for each 2D plane, and spectral windows in (t₁, t₂) of (1620, 7023) Hz, sampled over (128, 2210) points. Experiments performed at 14.1 T used ν_cpmg = (2 × 0, 50, 100, 2 × 300, 400, 500, 600, 700, 800, 900, 1000, and 1100) Hz, 2 s recovery delay, 24 scans for each 2D plane, and spectral windows in (t₁, t₂) of (2129, 9615) Hz, sampled over (128, 2306) points.