Identification of productive folding intermediates which account for the flow of protein folding pathway.

Reduced hirudin N-terminal fragment (Hir1-27, 4 cysteines) refolds to form two-disulfide structures. The isomer containing the native disulfide prevailed as the predominant product. The disulfide folding pathway was elucidated by trapping the intermediates with acid (4% trifluoroacetic acid). All six possible one-disulfide intermediates were detected to exist in equilibrium with molar ratio of approximately 1:1:1:0.4:0.4:0.18. These intermediates were purified and structurally characterized. They were also allowed to resume the folding by reconstituting into alkaline buffer in order to evaluate the productivity of individual intermediate along the pathway. These results demonstrate that the most productive intermediate that specifies the pathway flow is neither a well populated species nor the one that contains the native disulfides.