Fig. 5.

¹⁵N relaxation measurements for Lys NH₃⁺ and Arg N_εH groups (Esadze et al., 2016, 2011; Nguyen, Lokesh, Volk, & Iwahara, 2017). Symbols representing pulses are the same as those used in Fig. 2C. The delays τ_a = 2.7 ms and τ_b = 1.3 ms are used for Lys NH₃⁺ groups, whereas τ_a = 2.3 ms and τ_b = 2.7 ms are used for Arg N_εH groups. The ¹⁵N carrier position is set to ~33 pm for Lys NH₃⁺ groups and to ~85 ppm for Arg N_εH groups. The ¹H carrier position is set to the H₂O resonance. Quadrature detection in the t₁ domain was achieved using States-TPPI, incrementing the phase φ₁. Although the use of τ_b = 1.3 ms for Lys NH₃⁺ groups retains 4N_xH_zH_z terms, the impact of these terms is eliminated in other parts of these pulse sequences (Nguyen, Lokesh, et al., 2017). To maintain the in-phase single-quantum ¹⁵N coherence, the ¹H WALTZ composite pulses are used during the t₁ period. (A) ¹⁵N longitudinal relaxation measurement. A ¹H 180° pulse that does not affect H₂O resonance is applied every 10 ms during the delay T_r for longitudinal relaxation. A recycle delay between scans is set to 2 s in this experiment. Phase cycles: φ₁ = (2y, 2(−y)), φ₂ = (y, −y), φ₃ = (4x, 4(−x)), φ₄ = (8y, 8(−y)), and receiver = (x, −x, −x, x, 2(−x, x, x, −x), x, −x, −x, x). (B) ¹⁵N transverse relaxation measurement. The ¹H carrier position is shifted to 7.8 ppm at the position of the first vertical arrow (after the PFG g₄) and set back to the position of water resonance at the position of the second arrow. Typically we use the CPMG frequency ν_CPMG of 417 Hz. For this, the RF strength ω_CW/2π of ¹H CW during the CPMG is set to 5 kHz, satisfying ω_CW/2π = 2kν_CPMG (k, integer) (Hansen, Vallurupalli, & Kay, 2008). However, when a lower CPMG frequency is used, a stronger RF strength is required for ¹H CW to maintain the in-phase single-quantum ¹⁵N coherence during the CPMG scheme. For Arg N_εH groups, the CPMG frequency, the RF strength, and ¹⁵N carrier position should be set to avoid the recoupling conditions for modulation by ²J_NN couplings (Nguyen & Iwahara, 2018). The delays ξ₁ and ξ₂ are for alignment of ¹H magnetization and given by ξ₁ = 1/ω_CW = (4/π)τ_90H and ξ₂ = τ_90N − (2/π)τ_90H (Hansen et al., 2008; Hansen & Kay, 2007), in which τ₉₀ represents the relevant 90° pulse length. A recycle delay of 2.9 s is used. Phase cycles: φ₁ = (4y, 4(−y)), φ₂ = (8y, 8(−y)), φ₃ = x, φ₄ = (x, −x), φ₅ = (2y, 2 (−y)), φ₆ = (2x, 2(−x)), φ₇ = (2(−y), 2y), and receiver = (x, −x, x, −x, 2(−x, x, −x, x), x, −x, x, −x). (C) Heteronuclear ¹H–¹⁵N NOE measurement. Measurement with ¹H saturation (5 s) is performed with a train of 180°x and 180°(−x) pulses (RF strength, 11 kHz) at an interval of 10 ms. The reference spectrum is measured without the scheme in the bracket. For measurements of heteronuclear NOE, we set a recycle delay (including the saturation period) to 12s for a 600-MHz spectrometer and to 18s for 750 or 800 MHz spectrometer. Phase cycles: φ₁ = (y, −y), φ₂ = (4x, 4y, 4(−x), 4(−y)), φ₃ = (2x, 2(−x)), and receiver = (x, −x, −x, x, −x, x, x, −x).