A hierarchical pathway of protein folding can enable segmental unfolding by design. A monomeric insulin analogue containing pairwise substitution of internal A6-A11 cystine with serine [[Ser(A6),Ser(A11),Asp(B10),Lys(B28),Pro(B29)]insulin (DKP[A6-A11](Ser))] was previously investigated as a model of an oxidative protein-folding intermediate [Hua, Q. X., et al. (1996) J. Mol. Biol. 264, 390-403]. Its structure exhibits local unfolding of an adjoining amphipathic alpha-helix (residues A1-A8), leading to a 2000-fold reduction in activity. Such severe loss of function, unusual among mutant insulins, is proposed to reflect the cost of induced fit: receptor-directed restoration of the alpha-helix and its engagement in the hormone's hydrophobic core. To test this hypothesis, we have synthesized and characterized the corresponding alanine analogue [[Ala(A6),Ala(A11),Asp(B10),Lys(B28), Pro(B29)]insulin (DKP[A6-A11](Ala))]. Untethering the A6-A11 disulfide bridge by either amino acid causes similar perturbations in structure and dynamics as probed by circular dichroism and (1)H NMR spectroscopy. The analogues also exhibit similar decrements in thermodynamic stability relative to that of the parent monomer as probed by equilibrium denaturation studies (Delta Delta G(u) = 3.0 +/- 0.5 kcal/mol). Despite such similarities, the alanine analogue is 50 times more active than the serine analogue. Enhanced receptor binding (Delta Delta G = 2.2 kcal/mol) is in accord with alanine's greater helical propensity and more favorable hydrophobic-transfer free energy. The success of an induced-fit model highlights the applicability of general folding principles to a complex binding process. Comparison of DKP[A6-A11](Ser) and DKP[A6-A11](Ala) supports the hypothesis that the native A1-A8 alpha-helix functions as a preformed recognition element tethered by insulin's intrachain disulfide bridge. Segmental unfolding by design provides a novel approach to dissecting structure-activity relationships.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val A3 , which projects within a crevice between the A-and B-chains. Engineered receptor ␣-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap B25 -insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and ␣-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly A3 and Ser A3 ) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba A3 and allo-Thr A3 analogs are reduced at least 10-fold; the activity of Thr A3 -DKPinsulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba A3 ) or by altered polarity (allo-Thr A3 and Thr A3 ). Our results provide evidence that Val A3 , a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the ␣-subunit in a hormone-receptor complex.Binding of insulin to its receptor is central to the hormonal control of metabolism. Although recent crystallographic studies have provided insights into the structure of the free receptor ectodomain (1), mechanisms underlying hormone-receptor recognition remain elusive. Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues), derived from proinsulin (2). In the pancreatic ␤-cell insulin is stored as Zn 2ϩ -stabilized hexamers within secretory granules (2). The hexamers dissociate on secretion into the portal circulation, enabling the hormone to function as a Zn 2ϩ -free monomer. In this study we investigate structure-function relationships at position Val A3 , a site of mutation associated with diabetes mellitus (3) located within a crevice between A-and B-chains (Fig. 1A).Diabetes-associated mutations have been identified at three invariant sites as follows: Val A3 3 Leu A3 (insulin Wakayama), Phe B24 3 Ser (insulin Los Angeles), and Phe B25 3 Leu (insulin Chicago). These variants exhibit impaired receptor binding but to different extents (4). The most severely affected is Leu A3 -insulin, whose receptor binding is reduced by 500-fold. Such a marked impairment, unusual among mutant insulins (5-7), suggests either nonlocal structural perturb...