Fig. 4

NMR spectra of GY-23 peptide at different pH values (cross-peak trajectories marked with dashed lines). a ¹H-¹⁵N-HMQC spectrum at initial conditions of pH 3.3. b Overlay of ¹H-¹⁵N-HMQC spectra acquired between pH 3.3 and 7 (pH 7: initiation of LLPS). c, d Overlay of ¹H-¹³C-HSQC spectra of aliphatic (c) and aromatic (d) side chains at pH 3.3 and 7. The inset shows Tyr ¹H_δ-¹³C_ζ cross-peaks at pH 7. e Overlay of long-range ¹H-¹⁵N-HMQC spectra of His side chains. The resonance assignments in the protonated state (pH 3.3) are indicated. f Long-range ¹H-¹⁵N-HMQC spectrum at pH 7 acquired within 5 min after pH adjustment showing transient stabilization of His ε-tautomer with characteristic resonance at ca. 250 ppm marked with the arrow. In the spectrum acquired after 30 min of pH adjustment, this cross-peak was significantly attenuated (Supplementary Fig. 11). Spectra acquired at 298 K and peptide concentration of 1.5 mM. The trajectories (chemical shift values vs. pH) of ¹³C atoms of Tyr as well as ¹³C and ¹⁵N of His are provided in Supplementary Fig. 12.