The disulfide folding pathway of hirudin elucidated by stop/go folding experiments.

The folding pathway of hirudin was analyzed by structural characterization and stop/go folding experiments of acid-trapped intermediates. The results show that the folding is initiated by a near-random packing, followed by the reorganization and fine adjustment of partially compact intermediates to attain the active molecule. The process of packing is observed as the unfolded hirudin flows sequentially via three groups of equilibrated intermediates, namely one-disulfide, two-disulfide, and three-disulfide (scrambled species) isomers. Nearly all possible disulfide species were found to exist along the pathway. Specific tertiary interactions then take effect at the final stage, refining and consolidating the loosely packed intermediates, in the presence of free thiols, to form the active hirudin. The rate of packing and reorganization can be selectively regulated by a number of external factors, and conditions can be chosen to allow the completion of folding process within 10 min or 10 h.