Figure 4.

Selected regions of the 2D H^β/γ ₂(C^β/γ)C^γ/δ spectra of uniformly ¹⁵N/¹³C‐labeled T26H–T4L* in H₂O (blue; pH 5.8) and D₂O (red; pH* 5.7) NMR sample buffer. The spectra show correlations between (A) the H^ββ’ and ¹³C^γ of Asp and (B) the H^γγ’ and ¹³C^δ of Glu. The vertical insets are the 1D ¹³C‐NMR spectra of protein selectively labeled with (A) ¹³C^γ‐Asp or (B) ¹³C^δ‐Glu at pH 5.9 (taken from Fig. 3). The scale bar indicates the expected 0.23 ppm isotope shift for a neutral carboxylic acid (¹³COOH vs. ¹³COOD). The lack of any significant isotope shifts (< 0.04 ppm in magnitude) demonstrates that the Asp and Glu residues in T26H–T4L* are predominantly ionized under these conditions. Two exceptions are the small ¹³C^δ shift of 0.08 ppm exhibited by Glu11 (pK _a 4.7) that is attributed to a change in its fractional ionization due to the slightly different pH/pH* conditions, and the slight “reversed” isotope shift of Asp70 ¹³C^γ (−0.07 ppm) that may possibly result from protonation versus deuteration of its hydrogen bonded partner His31.22, 23 Also seen in (A) are ¹⁵N‐coupled signals from several Gln sidechains, which show expected20, 24 deuterium isotope shifts (¹³CONH₂ vs. ¹³COND₂) of ~ 0.12 ppm. Signals from the Asn sidechains, with similar isotope shifts, are outside of the presented ¹³C chemical shift windows.