A Major Kinetic Trap for the Oxidative Folding of Human Epidermal Growth Factor

Jui-Yoa Chang,Li Li,Por-Hsiung Lai

doi:10.1074/jbc.m005160200

Abstract

The folding pathway of human epidermal growth factor (EGF) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. Oxidative folding of the fully reduced EGF proceeds through 1-disulfide intermediates and accumulates rapidly as a single stable 2-disulfide intermediate (designated as EGF-II), which represents up to more than 85% of the total protein along the folding pathway. Among the five 1-disulfide intermediates that have been structurally characterized, only one is native, and nearly all of them are bridges by neighboring cysteines. Extensive accumulation of EGF-II indicates that it accounts for the major kinetic trap of EGF folding. EGF-II contains two of the three native disulfide bonds of EGF, Cys(14)-Cys(31) and Cys(33)-Cys(42). However, formation of the third native disulfide (Cys(6)-Cys(20)) for EGF-II is slow and does not occur directly. Kinetic analysis reveals that an important route for EGF-II to reach the native structure is via rearrangement pathway through 3-disulfide scrambled isomers. The pathway of EGF-II to attain the native structure differs from that of three major 2-disulfide intermediates of bovine pancreatic trypsin inhibitor (BPTI). The dissimilarities of folding mechanism(s) between EGF, BPTI, and hirudin are discussed in this paper.

Highlights

The folding pathway of human epidermal growth factor (EGF) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates
2) Scrambled 3-disulfide isomers, which have not been observed in the case of bovine pancreatic trypsin inhibitor (BPTI), were shown to serve as folding intermediates of hirudin, potato carboxypeptidase inhibitor (PCI), as well as tick anticoagulant peptide
The folding intermediates of EGF [14] consist of scrambled isomers that are not found in the case of BPTI and predominant 2-disulfide species that are absent from the pathway of hirudin

Summary

A Major Kinetic Trap for the Oxidative Folding of Human Epidermal Growth Factor*

Using the same technique of acid trapping, our laboratory has analyzed the folding pathway(s) of three single domain, 3-disulfide containing proteins that have sizes similar to that of BPTI, including hirudin [10], potato carboxypeptidase inhibitor (PCI) [11], and tick anticoagulant peptide [12] Their folding mechanism(s) have been shown to differ from that of BPTI in two important aspects. The folding intermediates of EGF [14] consist of scrambled isomers that are not found in the case of BPTI and predominant 2-disulfide species that are absent from the pathway of hirudin These discrepancies indicate that the folding pathway of small disulfide containing proteins is more complex than what has been learned from the BPTI model alone. Kinetic analysis of a predominant 2-disulfide intermediates of EGF using the stop/go folding experiments

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

A Major Kinetic Trap for the Oxidative Folding of Human Epidermal Growth Factor