Refolding mechanism of ovalbumin: investigation by using a starting urea-denatured disulfide isomer with mispaired CYS367-CYS382.
Ovalbumin, a member of the serpin superfamily, contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state. To investigate the folding mechanism of ovalbumin, a urea-denatured disulfide isomer with a mispaired disulfide Cys367-Cys382 (D[367-382]) and its derivative (D[367-382/CM-73]) in which a native cystine counterpart of Cys73 is blocked by carboxymethylation were produced. Both the denatured isomers refolded within an instrumental dead time of 4 ms into an initial burst intermediate IN with partially folded conformation. After the initial burst phase, most of the D[367-382] molecules further refolded into the native form. In contrast, upon dilution of D[367-382/CM-73] with the refolding buffer, the protein stayed in the IN state as a stable form, which displayed a partial regain of the native secondary structure and a compact conformation with a similar Stokes radius to the native form. The structural characteristics of IN were clearly differentiated from those of an equilibrium intermediate IA that was produced by dilution with an acidic buffer of urea-denatured ovalbumin; IA showed much more hydrophobic dye binding and a larger Stokes radius than the IN state, despite their indistinguishable far-UV circular dichroic spectra. The non-productive nature of IA highlighted the importance of a compact conformation of the IN state for subsequent native refolding. These observations were consistent with a refolding model of ovalbumin that includes the regain of the partial secondary structure and of the compactness of overall conformation in an initial burst phase before the subsequent native refolding.
Onda
Maki
M
Department of Environmental Sciences, Faculty of Science, Osaka Women's University, Daisen-cho 2-1, Sakai, Osaka 590-0035, Japan.
Hirose
Masaaki
M
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D003553:Cystine; D007536:Isomerism; D007700:Kinetics; D010047:Ovalbumin / Q000737:chemistry*; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D014508:Urea / Q000494:pharmacology
D003553:Cystine; D014508:Urea; D010047:Ovalbumin