Structural and functional characteristics of partially disulfide-reduced intermediates of ovotransferrin N lobe. Cystine localization by indirect end-labeling approach and implications for the reduction pathway.
Ovotransferrin N lobe contains six intrachain disulfides (SS-I/Cys10-Cys45; SS-II/Cys20-Cys36; SS-III/Cys115-Cys197; SS-IV/Cys160-Cys174; SS-V/Cys171-Cys182; SS-VI/Cys228-Cys242) in a single polypeptide chain of 332 amino acid residues. Upon the protein disulfide reduction with dithiothreitol under nondenaturing conditions, the intermediate species with four, three, and two disulfides were generated. The partially disulfide-reduced intermediates were isolated, and the localization of intact disulfides in the intermediates was determined by an indirect end-labeling method. This method included the S-cyanocysteine-specific protein fragmentation, followed by gel electrophoresis and the immunochemical visualization of the C terminus-intact fragments using antiserum raised against a non-cysteine C-terminal fragment (Ser280-Arg332). Results clearly showed that first SS-IV and SS-V, second SS-III, and then SS-VI are cleaved. No reduction was observed for SS-I and SS-II under the employed reducing conditions. The conclusion was confirmed by peptide mapping analyses for the same disulfide intermediates using reverse phase high performance liquid chromatography. Transverse urea gradient gel electrophoresis and visible absorption spectra revealed that the four-disulfide intermediate, but not the three- or two-disulfide intermediate, retains essentially the same iron-binding function as the native protein. By far-UV CD analyses, the residual native conformation of the partially disulfide-reduced intermediates was found to decrease with increased number of the reduced disulfides. Implications of the partially disulfide-reduced intermediates for the disulfide-reductive unfolding pathway in ovotransferrin N lobe are discussed.
Yamashita
H
H
Research Institute for Food Science, Kyoto University, Japan.
Nakatsuka
T
T
Hirose
M
M
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003207:Conalbumin / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008961:Models, Structural; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D013053:Spectrophotometry
D004220:Disulfides; D010446:Peptide Fragments; D003207:Conalbumin; D003545:Cysteine