(A) 1H-15N HSQC spectra acquired on a 200 µM sample of HSPB5-ACD at pH 7.5 and 6.5 (22°C) reveal two states. Full spectra collected at pH 7.5 (black) and 6.5 (red) are overlaid (left). At pH 7.5, 82/85 non-proline residues are observed (residues G64, L65, and S139 are not detected due to fast exchange with H2O). At pH 6.5, approximately ∼65 additional peaks appear, indicating the presence of two conformations. Boxed regions shown on the right provide clear examples of peak doubling. Some resonances (labeled in the panel on the left) disappear from their original positions at pH 7.5 and their new positions could not be determined by inspection of the spectrum at pH 6.5. (B) Example of a full pH titration series (spectra collected at pH 8.4 [blue], 7.5 [black], 7.0 [cyan], 6.7 [green], and 6.5 [red]). The behavior of the resonance of H119 as a function of pH illustrates two pH-dependent processes (see text). Its chemical shift is in fast exchange from pH 8.4 to 7.5 (solid arrow) and changes direction and is in slow exchange from pH 7.0 to 6.5 (dashed arrow). The region shown contains several other resonances that undergo slow-exchange transitions over the same pH range. (C) Residues that undergo the slow exchange transition are highlighted in color on a surface representation of the ACD dimer. Residues with perturbations > 0.2 ppm are red; those with perturbations between 0.1 - 0.2 ppm are orange. (D) Analysis of relaxation dispersion data yields the difference in 15N chemical shift between the major and minor species (ΔδNcalc). The values of ΔδNcalc (blue) are compared to the experimental values obtained from the difference of 15N chemical shifts at pH 7.5 and 6.5, i.e., ΔδN(7.5−6.5) (red), in the histogram. Concordance between these two parameters supports the notion that the minor form detected by relaxation dispersion experiments is the monomeric form of ACD that is populated at lower pH.