Figure 1.

Comparison between 2D HyperW (red) and conventional (blue) ¹H–¹⁵N HMQC spectra measured on ¹⁵N-PhoA4. 2.8 mL of super-heated buffered D₂O (50 mM HEPES, pD 7.5, 50 mM KCl) was used to dissolve an 85/15 water/glycerol pellet containing 10 mM 4-amino-TEMPO. This pellet had been polarized at 1.12 K for ∼3 h 30 min using 100 mW of microwave irradiation at 94.195 GHz. ∼240 μL of the resulting hyperpolarized water solutions were injected into a 5 mm NMR tube containing ∼140 μL of a 1.5 mM ¹⁵N-labeled PhoA4 solution. Partial tentative assignment of residues indicated by single-letter amino acid codes is done based on the BMRB entry of the full-length PhoA.³² Both spectra were recorded at 50 °C using 64 hypercomplex t₁ increments and hypercomplex³⁴ acquisition covering indirect- and direct-domain spectral widths of 6009.6 and 1825.8 Hz. The HyperW spectrum was recorded using two phase-cycled scans per t₁ increment. Additional experimental parameters: 14.1 T Prodigy-equipped NMR; total acquisition times of 73 s for the hyperpolarized spectrum (acquisition time of 213.0 ms, repetition delay of 0.037 s) and 14 h 12 min for the thermal spectrum (320 scans per t₁ increment, acquisition time of 213.0 ms, and a repetition delay of 1 s).