Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide.
Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33-Cys55). Two major two-disulfide isomers were TAP-(Cys33-Cys55, Cys15-Cys39) and TAP-(Cys33-Cys55, Cys5-Cys39). Both Cys33-Cys55 and Cys15-Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30-Cys51), BPTI-(Cys30-Cys51, Cys14-Cys38), and BPTI-(Cys30-Cys51,Cys5-Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.
Chang
J Y
JY
Research Center for Protein Chemistry, Institute of Molecular Medicine, University of Texas, Houston 77030, USA. rchang@imm2.imm.uth.tmc.edu
Ballatore
A
A
D000595:Amino Acid Sequence; D060829:Arthropod Proteins; D004220:Disulfides / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding
D060829:Arthropod Proteins; D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C063967:tick anticoagulant peptide