Size of hirudin sequence required to fold into an active core domain.
The active core domain of hirudin contains three native disulfides (Cys6-Cys14,Cys16-Cys28, and Cys22-Cys39) and 49 amino acid residues (Hir1-49). This compact structure folds spontaneously, and its folding pathway has been elucidated recently [Chatrenet and Chang (1993) J. Biol. Chem. 268, 20988-20996]. The folding mechanisms of Hir1-35 and Hir1-43 were investigated in order to determine the minimum structural elements required to fold into this active core structure. Hir1-35 includes two native disulfides (Cys6-Cys14 and Cys16-Cys28) and an extra Cys22. When reduced/denatured Hir1-35 was allowed to fold, it folded into a collection of equilibrated 2-disulfide isomers. At least eight fractions of the 2-disulfide species have been observed. Structural analysis revealed that out of the 10 possible disulfide pairings, only five were detected to exist in the 2-disulfide isomers, and all have their half-cystines separated by less than 8-10 amino acid residues. One of the native disulfides, Cys16-Cys28, has not been found in any of those 2-disulfide species. On the other hand, the C-terminal extension of an octapeptide permitted reduced/denaturated Hir1-43 to fold into a defined active structure possessing the three native disulfides. These results also demonstrate that the folding mechanism of Hir1-35 resembles what occurs during the early stage of Hir1-43 (and Hir1-49) folding, which involves a process of nonspecific packing of unfolded polypeptide chains.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding
D004220:Disulfides; D006629:Hirudins; D010446:Peptide Fragments