Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate.
The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.
Ke
Huimin
H
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Zhang
Sen
S
Li
Jian
J
Howlett
Geoffrey J
GJ
Wang
Chih-chen
CC
D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D033903:Periplasmic Proteins; D019704:Protein Disulfide-Isomerases