Figure 4.

Helices H0/H1 are marginally stable with little protection from HX. Amide HX rate constants were determined from CLEANEX experiments recorded at 25°C for (A) PntP2^142–252 (pH 6.7 and pH 7.5), (B) 2P-PntP2^142–252 (pH 6.7), and (C) 3P-PntP2^142–252 (pH 7.1 and 7.5), and normalized to pH 6.7, assuming a first-order dependence on [OH–]. For better comparison, the bars for several data points were truncated and the rate constants were indicated by the numbers. Phosphothreonines are identified with asterisks. Missing data points correspond to prolines, amides with overlapping ¹⁵N-HSQC signals, and amides with HX rates too slow to be measured by the CLEANEX approach under the sample pH conditions examined (i.e., k_ex > 0.5 s⁻¹). Most amides fall in the latter category owing to their presence in stable, hydrogen-bonded structural elements of the protein. However, in the case of 3P-PntP2^142–252, the three phosphothreonines likely exchange rapidly but were not included in (C) owing to ambiguous spectral assignments at elevated sample pH values.