Isomers of Epidermal Growth Factor with Ser ⇒ Cys Mutation at the N-Terminal Sequence:  Isomerization, Stability, Unfolding, Refolding, and Structure

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys6−Cys20, Cys14−Cys31, and Cys33−Cys42. The connection of Cys6 and Cys20 forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738−1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845−4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser ⇒ Cys mutation at Ser residues (Ser2, Ser4, and Ser9) flanking Cys6. This allows competition between Cys6 and mutated Cys2, Cys4, and Cys9 to link with Cys20 and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys6−Cys20 is favored over Cys2−Cys20 and Cys4−Cys20 by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys9−Cys20 is thermodynamically more stable than the native Cys6−Cys20 by a free-energy difference (ΔG°) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.