Fig. 2.

The importance of maintaining the in-phase single-quantum term during the ¹⁵N evolution period in ¹H–¹⁵N NMR experiments for Lys and Arg side chains. (A) Impact of scalar relaxation on different single-quantum terms of ¹⁵N coherence. (B) Broadening of ¹⁵N line shapes due to hydrogen exchange in the ¹⁵N dimensions of HSQC, HMQC, and HISQC spectra for Lys NH₃⁺ and Arg N_εH groups. The increases in ¹⁵N half-height widths are given in Hz units. (C) The pulse sequence of the water-flip-back HISQC experiment for Lys NH₃⁺ or Arg N_εH groups. Thin and bold bars in black represent hard rectangular 90° and 180° pulses, respectively. Water-selective half-Gaussian (2.1 ms) and soft-rectangular (1.2 ms) 90° pulses are represented by half-bell and short-bold shapes, respectively. Unless indicated otherwise, pulse phases are along x, and the carrier position for ¹H was set to the position of the water resonance. The ¹⁵N carrier position is set to ~33 ppm for Lys NH₃⁺ groups and to ~85 ppm for Arg N_εH groups. A gray bell-shape for ¹⁵N represents a 1.0-ms rSNOB 180° pulse (Kupče, Boyd, & Campbell, 1995) selective to Lys ¹⁵N_ζ or Arg ¹⁵N_ε nuclei. The delays τ_a and τ_b are set as follows: τ_a = 2.7 ms and τ_b = 1.3 ms for Lys NH₃⁺ groups; τ_a = 2.3 ms and τ_b = 2.7 ms for Arg N_εH groups. This setting for Arg side chains suppresses signals from N_ηH₂ groups; τ_b = 1.3 ms can be used to avoid this suppression of N_ηH₂ signals. Phase cycles: φ₁ = (y, −y); φ₂ = (2(y), 2(−y)); φ₃ = (4x, 4(−x));receiver = (x, 2(−x), x, −x, 2x, −x). Quadrature detection in the t₁ domain was achieved using States-TPPI, incrementing the phase φ₁. The third ¹H 90° pulse along −x (before the gradient g₄) serves two purposes: it purges unnecessary ¹⁵N antiphase magnetization by generating multiple quantum coherence and it flips the water magnetization back to +z. In the HISQC experiment, ¹H WALTZ-16 decoupling pulses maintain ¹⁵N in-phase single-quantum coherence during the ¹⁵N evolution period t₁ and thereby suppress the broadening of ¹⁵N line shapes due to rapid scalar relaxation arising from hydrogen exchange. (D) HISQC drastically improves ¹H–¹⁵N correlation spectra for Lys NH₃⁺ groups through suppression of scalar relaxation during the ¹⁵N evolution period. This panel compares the HMQC, HSQC, and HISQC spectra recorded for the Lys NH₃⁺ groups of the ¹⁵N-labeled Egr-1 zinc-finger–DNA complex at pH 5.8 and 10°C. For each spectrum, the data were acquired using the same number of scans and the same digital resolutions and were processed identically. F₁-slices at the position indicated by an arrow are shown to indicate ¹⁵N line shapes.