Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor.
The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.
Salamanca
Silvia
S
Institut de Biotecnologia i Biomedicina and Department de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Li
Li
L
Vendrell
Josep
J
Aviles
Francesc X
FX
Chang
Jui-Yoa
JY
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000737:chemistry; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
D004220:Disulfides; D006629:Hirudins; C116332:LCI protein, Hirudo medicinalis; D011506:Proteins; D007611:Aprotinin; D003545:Cysteine