How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding D and L amino acid substitutions in the hormoneto define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly B8 lies within a -turn and exhibits a positive ϕ angle (like a D amino acid); in the alternative R state, Gly B8 is part of an -helix and exhibits a negative ϕ angle (like an L amino acid). Respective B chain libraries containing mixtures of D or L substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: L amino acids impede native disulfide pairing, whereas diverse D substitutions are well-tolerated. Strikingly, D substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, L analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of D and L analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 ϕ angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated † This work was supported in part by Diabetes Research and Training Center at the University of Chicago (S. H SUPPORTING INFORMATION AVAILABLENine figures illustrating disulfide pairing and structural relationships in insulin crystals, visible absorption spectra of cobalt-substituted hexamers, additional CD and NMR spectra, diagonal plot of NOEs, and summary of NMR sequential assignment. Nine tables providing B8 dihedral angles, summary of mutations at sites neighboring B8, crystallographic unit-cell dimensions, NMR resonance assignments, statistical information pertaining to DG/RMD ensemble, and restraints. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2013 December 02. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assemb...

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.