Figure 7.

SH3 folded and unfolded states visualized by HyperW HMQC. (A) 2D ¹H–¹⁵N HMQC spectra measured for a ∼520 μM ¹⁵N-drkN-SH3 domain in an 87.4% H₂O buffer (50 mM HEPES buffer, 50 mM KCl, pH 7.5) at 27 °C (blue), 37 °C (green), and 50 °C (black). Indirect- and direct-domain spectral widths of 9014.4 and 2312.7 Hz were covered, using 64 hypercomplex t₁ increments.³⁴ The flip angle of the selective excitation was 90°, and 16 scans were collected using a 56.8 ms acquisition time and a relaxation delay of 2 s. Total experimental time was 1 h 12 min. (B) Enlarged region of Trp36 indole peak (marked with a black rectangle in (A)) showing the thermally driven rise of the unfolded state. (C) Comparison between 2D HyperW (red) and thermally polarized (blue) ¹ H–¹⁵N HMQC spectra measured for the ¹⁵N-drkN-SH3 domain. 2.8 mL of 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. The pellet was polarized at 1.17 K for 3 h 30 min using 100 mW microwave irradiation at 94.195 GHz. ∼180 μL of the resulting hyperpolarized water solution was injected into a 5 mm NMR tube containing ∼140 μL of a ∼1.3 mM ¹⁵N-drkN-SH3 solution. Partial assignment of the various residues indicated by single-letter amino acid codes is done on the basis of Zhang et al.⁶⁵ Resonances of the folded conformation are labeled with these assignments, and resonances of the unfolded form are marked with an added prime (′). These spectra were recorded at 50 °C using 64 hypercomplex t₁ increments covering indirect- and direct-domain spectral widths of 7211.5 and 1947.5 Hz. The HyperW spectrum was recorded using two phase-cycled scans per t₁ increment. Total experimental time was 63 s for the hyperpolarized spectrum (acquisition time of 177.5 ms, repetition delay of 0.037 s) and 13 h 50 min for the thermal spectrum (176 scans, acquisition time of 177.5 ms and a repetition delay of 2 s per t₁ increment). (D) Enlarged region of the Trp36 indole peak (marked with a black rectangle in (C)).