Hierarchical Protein Folding: Asymmetric Unfolding of an Insulin Analogue Lacking the A7−B7 Interchain Disulfide Bridge

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Abstract
The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (CysA7−CysB7) are characterized as a model of a late intermediate. Untethering cystine A7−B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6−A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry39, 15429−15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20−B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains nativelike supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.