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Size of hirudin sequence required to fold into an active core domain
1Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Insights
Investigating hirudin folding, researchers found that Hir1-35 forms multiple disulfide isomers, while Hir1-43 with an octapeptide extension folds correctly. This reveals minimal structural requirements for active hirudin core folding.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Folding
Background:
- The active core domain of hirudin (Hir1-49) contains three native disulfides and folds spontaneously.
- Previous studies have elucidated the folding pathway of the full hirudin core.
Purpose of the Study:
- To determine the minimum structural elements required for the active core structure of hirudin to fold.
- To investigate the folding mechanisms of hirudin fragments Hir1-35 and Hir1-43.
Main Methods:
- Investigated folding of hirudin fragments Hir1-35 and Hir1-43.
- Analyzed disulfide bond formation and structural integrity of folded fragments.
- Compared folding pathways of different hirudin constructs.
Main Results:
- Hir1-35, with two native disulfides and an extra Cys22, folded into multiple 2-disulfide isomers, with five out of ten possible pairings detected.
- The native disulfide Cys16-Cys28 was absent in Hir1-35 2-disulfide species.
- The C-terminal octapeptide extension in Hir1-43 enabled folding into a defined active structure with three native disulfides.
Conclusions:
- The folding of Hir1-35 mimics early-stage folding of Hir1-43/Hir1-49, involving nonspecific polypeptide chain packing.
- Specific structural elements, including the C-terminal region, are crucial for achieving the native hirudin active core structure.
Abstract:
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.