FIGURE 4.
Upfield region of 750-MHz one-dimensional 1H NMR spectra of cDsbD and nDsbD show oxidation state-dependent interactions. a, titration of C461A-cDsbD, mimicking cDsbDred, with nDsbDox shows slow exchange on the NMR time scale. Stepwise additions of a 1.2 mm stock solution of nDsbDox to a ∼0.3 mm solution of C461A-cDsbD were made. nDsbD concentrations were 0.11, 0.15, and 0.22 mm. As nDsbD is added, the peak corresponding to Ala458 broadens and decreases in intensity but does not shift. A new peak at −0.77 ppm corresponding to Ala458 in the nDsbD-bound state appears and increases in intensity. b, nDsbDox was titrated into a mixture of ∼0.3 mm C461A-cDsbD and ∼0.3 mm cDsbDox. As the concentration of nDsbDox increases from 0.015 to 0.043 mm, significant broadening is observed for the Ala458 peak of free C461A-cDsbD. The peak corresponding to Ala458 in wild-type cDsbDox is not observed to shift or broaden. This confirms that nDsbDox interacts preferentially with C461A-cDsbD. c, nDsbDox was titrated into a mixture of 0.2 mm cDsbDox and 0.2 mm V462A-cDsbDox. As the concentration of nDsbDox increases from 0.056 to 0.107 mm, the peak corresponding to Ala458 of wild-type DsbDox broadens and shifts upfield, whereas the peak corresponding to Ala458 in V462A-cDsbDox is not perturbed. This indicates that nDsbDox interacts preferentially with wild-type cDsbD. d, nDsbDox was titrated into a mixture of 0.25 mm cDsbDox and 0.25 mm D455N/E468Q-cDsbDox. As the concentration of nDsbDox increases from 0.082 to 0.144 mm, the peak corresponding to Ala458 of wild-type DsbDox broadens and shifts upfield to a more significant extent than the peak corresponding to Ala458 in D455N/E468Q-cDsbDox. This indicates that nDsbDox has a higher affinity for wild-type cDsbDox than for D455N/E468Q-cDsbDox.