cDsbDox interacts more weakly with nDsbDred than with nDsbDox. a, two-dimensional 1H-15N HSQC spectrum of 0.10 mm [15N]cDsbDox. b, HSQC spectrum obtained after the addition of 0.04 mm C109A-nDsbD, mimicking nDsbDred, to 0.10 mm [15N]cDsbDox. Peaks arising from Val462 and Leu508 show significant broadening. c and d, HSQC spectrum obtained after the addition of 0.010 mm (c) and 0.014 mm (d) nDsbDox to 0.10 mm [15N]cDsbDox. The peaks corresponding to Val462 and Leu508 in b show a level of broadening that is intermediate between that observed in c and d. e, a comparison of the upfield region of 750-MHz one-dimensional 1H NMR spectra obtained for the samples shown in b (lower trace) and d (upper trace). This demonstrates that a higher concentration of C109A-nDsbD is required to achieve the same degree of complex formation compared with nDsbDox.