title: Hierarchical protein folding: asymmetric unfolding of an insulin analogue lacking the A7-B7 interchain disulfide bridge.
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 (Cys(A7)-Cys(B7)) 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) Biochemistry 39, 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 native-like 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44016, USA.
authors.forename: Q X
authors.initials: QX
authors.lastname: Hua
authors.forename: S H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: W
authors.initials: W
authors.lastname: Jia
authors.forename: S Q
authors.initials: SQ
authors.lastname: Hu
authors.forename: Y C
authors.initials: YC
authors.lastname: Chu
authors.forename: P G
authors.initials: PG
authors.lastname: Katsoyannis
authors.forename: M A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004355:Drug Stability; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011384:Proinsulin / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D007328:Insulin; D021122:Protein Subunits; D011384:Proinsulin