Characterizing the Tick Carboxypeptidase Inhibitor MOLECULAR BASIS FOR ITS TWO-DOMAIN NATURE*□S

Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI con-sists of two domains that are structurally very similar, each con-taining three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding path-ways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential for-mation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two pre-dominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumu-late along the reaction and are strongly prevented by the pres-ence of protein disulfide isomerase. This study demonstrate