title: Different roles of the two disulfide bonds of the cysteine proteinase inhibitor, chicken cystatin, for the conformation of the active protein.
abstract: The Cys-71-Cys-81 disulfide bond of the cysteine proteinase inhibitor, chicken cystatin, was specifically reduced by thioredoxin or low concentrations of dithiothreitol. This cleavage, followed by S-carbamoylmethylation, induced a conformational change of the protein, as evidenced by changes in isoelectric point and circular dichroism spectra and by an increased susceptibility to digestion by nontarget proteinases. The proteinase binding ability and the immunological properties of the inhibitor, however, were not detectably altered, indicating that the conformational change was limited to the region around the disrupted bond. In contrast, reduction of both disulfide bonds of cystatin by higher concentrations of dithiothreitol and subsequent alkylation led to the slow conversion of the inhibitor into two forms lacking proteinase binding ability, indicative of more extensive conformational changes. Together, these results suggest that the less accessible Cys-95-Cys-115 disulfide bond of chicken cystatin, but not the more accessible Cys-71-Cys-81 bond, is of importance for maintaining the conformation of the inhibitor required for binding of target proteinases.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
authors.forename: I
authors.initials: I
authors.lastname: Björk
authors.forename: K
authors.initials: K
authors.lastname: Ylinenjärvi
mesh_terms: D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003553:Cystine / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D007526:Isoelectric Point; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
chemical_list: D015891:Cystatins; D003553:Cystine; D013879:Thioredoxins; D003545:Cysteine; D004229:Dithiothreitol