Figure 5.

H/D exchange behavior of RH and RHΔNT. (A) ¹H-¹⁵N HSQC spectrum of RHmnel obtained during the first 40-min period after replacement of the H₂O buffer with D₂O buffer (black spectrum), overlaid with a spectrum obtained during the 40-min period starting at 6.6 h after buffer replacement (red spectrum). (B) ¹H-¹⁵N HSQC spectra of RHΔNT obtained during the first 40-min accumulation period after buffer exchange (black spectrum) and the second 40-min period (green spectrum). The amides protons of the cleavage site residues F440 and Y441 in RHmnel are readily observed after 6.6 h, while in RHΔNT they have been fully exchanged. Exchange studies were performed at 25 °C in 25 mM Tris-HCl-d11 in D₂O (pD 7.5), 100 mM KCl.