Role of native disulfide bonds in the structure and activity of insulin-like growth factor 1: genetic models of protein-folding intermediates.

Insulin and insulin-related proteins contain three motif-specific disulfide bonds. Here we examine the role of these disulfide bonds in the folding and function of one family member, human insulin-like growth factor 1 (IGF-1). Analogues containing pariwise Cys-->Ser or Cys-->Ala substitutions were expressed in Escherichia coli, purified, and analyzed with respect to receptor-binding, solution structure, and thermodynamic stability. An analogue lacking all three disulfide bonds (designated des-Cys-IGF-1) is inactive and unfolded. Introduction of the [18-61] disulfide bond, previously shown to occur in an early intermediate in oxidative refolding [Miller, J. A., Owers-Narhi, L., Hua, Q. X., Rosenfeld, R., Arakawa, T., Rohde, M., Prestrelski, S., Lauren, S., S. Stoney, K. S., Tsai, L., & Weiss, M. A. (1993) Biochemistry (preceding paper in this issue)], results in a compact partially folded state with low but significant biological activity. Additional but incomplete structural organization and biological activity are observed following introduction of either the [6-48] or the [47-52] disulfide bonds. Native function, structure, and stability require the presence of all three disulfide bonds. These analogues provide genetic models of IGF-1 protein-folding intermediates. Their characterization suggests that bifurcation of the IGF-1 folding pathway reflects alternative late steps in the folding of a molten-globule intermediate.