Hierarchical Protein “Un-Design”:  Insulin's Intrachain Disulfide Bridge Tethers a Recognition α-Helix

Abstract

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 Different Energetic State of the Intra A-Chain/Domain Disulfide of Insulin and Insulin-like Growth Factor 1 Is Mainly Controlled by Their B-Chain/Domain

“…The architecture of insulin and IGF-1 mainly consists of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; 8-18, 42-49, and 54-61 in IGF-1) in the A-and B-chain/domains ( Figure 1A,B). The three R-helical segments are stabilized by the three disulfides (A6-A11, A7-B7, A20-B19 in insulin; [47][48][49][50][51][52]. The conformation of the C-and D-domains of IGF-1 is highly flexible.…”

“…The three-dimensional structure of IGF-1 is very similar with that of insulin (7). The architecture of insulin and IGF-1 mainly consists of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; [8][9][10][11][12][13][14][15][16][17][18][42][43][44][45][46][47][48][49], and 54-61 in IGF-1) in the A-and B-chain/domains ( Figure 1A,B). The three R-helical segments are stabilized by the three disulfides (A6-A11, A7-B7, A20-B19 in insulin; 47-52, 6-48, 18-61 in IGF-1) (8)(9)(10).…”

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

“…The refolding of fully reduced [1][2][3][4][5][6][7][8][9][10]mini-IGF-1 produced only one product, which had a retention time identical to that of the intact [1][2][3][4][5][6][7][8][9][10]mini-IGF-1. disulfide results in unfolding of helix 2 in both insulin (31)(32)(33) and IGF-1 (34). So, the disulfide stability of A7-B7 and A6-A11 determine the stability of helix 2.…”

Proinsulin and the Genetics of Diabetes Mellitus

“…These bridges (A6-A11, A7-B7, and A20-B19) (gold in Fig. 1B) are essential for stability and bioactivity (17,(21)(22)(23)(24)(25)(26)(27)(28)(29). Proinsulin consists of a folded insulin-like moiety and disordered connecting peptide (the C-domain) (dashed black line in Fig.…”

“…This bridge, which packs within a cluster of conserved aliphatic and aromatic side chains in the hydrophobic core, is proposed to contribute to stabilization of a specific folding nucleus (17,59,60). The structural role of cystine A20-B19 in populated one-and two-disulfide intermediates has been investigated through construction of analogs containing pairwise substitution of the other cysteines with Ala or Ser (17,(21)(22)(23)(24)(25)(26)(27)(28)(29). Such analogs exhibit partial folds with attenuated but non-negligible ␣-helix content.…”